@article {pmid38567730, year = {2024}, author = {Skutel, M and Yanovskaya, D and Demkina, A and Shenfeld, A and Musharova, O and Severinov, K and Isaev, A}, title = {RecA-dependent or independent recombination of plasmid DNA generates a conflict with the host EcoKI immunity by launching restriction alleviation.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae243}, pmid = {38567730}, issn = {1362-4962}, support = {14-00004//RSF/ ; 075-10-2021-114//Ministry of Science and Higher Education/ ; //Skoltech Systems Biology Program/ ; //Institute of Science and Technology/ ; }, abstract = {Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis.}, }
@article {pmid38331210, year = {2024}, author = {Amundsen, SK and Smith, GR}, title = {Chi hotspot control of RecBCD helicase-nuclease: Enzymatic tests support the intramolecular signal-transduction model.}, journal = {Journal of molecular biology}, volume = {}, number = {}, pages = {168482}, doi = {10.1016/j.jmb.2024.168482}, pmid = {38331210}, issn = {1089-8638}, abstract = {Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5'-GCTGGTGG-3'). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model - upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes.}, }
@article {pmid38261972, year = {2024}, author = {Pavankumar, TL and Wong, CJ and Wong, YK and Spies, M and Kowalczykowski, SC}, title = {Trans-complementation by the RecB nuclease domain of RecBCD enzyme reveals new insight into RecA loading upon χ recognition.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae007}, pmid = {38261972}, issn = {1362-4962}, support = {R35 GM131900/NH/NIH HHS/United States ; }, abstract = {The loading of RecA onto ssDNA by RecBCD is an essential step of RecBCD-mediated homologous recombination. RecBCD facilitates RecA-loading onto ssDNA in a χ-dependent manner via its RecB nuclease domain (RecBn). Before recognition of χ, RecBn is sequestered through interactions with RecBCD. It was proposed that upon χ-recognition, RecBn undocks, allowing RecBn to swing out via a contiguous 70 amino acid linker to reveal the RecA-loading surface, and then recruit and load RecA onto ssDNA. We tested this hypothesis by examining the interactions between RecBn (RecB928-1180) and truncated RecBCD (RecB1-927CD) lacking the nuclease domain. The reconstituted complex of RecB1-927CD and RecBn is functional in vitro and in vivo. Our results indicate that despite being covalently severed from RecB1-927CD, RecBn can still load RecA onto ssDNA, establishing that RecBn does not function while only remaining tethered to the RecBCD complex via the linker. Instead, RecBCD undergoes a χ-induced intramolecular rearrangement to reveal the RecA-loading surface.}, }
@article {pmid38081382, year = {2023}, author = {Fazio, N and Mersch, KN and Hao, L and Lohman, TM}, title = {E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity.}, journal = {Journal of molecular biology}, volume = {}, number = {}, pages = {168381}, doi = {10.1016/j.jmb.2023.168381}, pmid = {38081382}, issn = {1089-8638}, abstract = {Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB[ΔNuc]CD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecB[D1080A]CD), which retains the nuclease domain, showed no change ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤ 60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecB[ΔNuc]CD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecB[ΔNuc]CD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecB[D1080A]CD unwinding are intermediate between RecBCD and RecB[ΔNuc]CD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecB[ΔNuc]CD may mimic a post-chi state of RecBCD.}, }
@article {pmid38047637, year = {2023}, author = {Amundsen, SK and Smith, GR}, title = {RecBCD enzyme: mechanistic insights from mutants of a complex helicase-nuclease.}, journal = {Microbiology and molecular biology reviews : MMBR}, volume = {}, number = {}, pages = {e0004123}, doi = {10.1128/mmbr.00041-23}, pmid = {38047637}, issn = {1098-5557}, abstract = {SUMMARYRecBCD enzyme is a multi-functional protein that initiates the major pathway of homologous genetic recombination and DNA double-strand break repair in Escherichia coli. It is also required for high cell viability and aids proper DNA replication. This 330-kDa, three-subunit enzyme is one of the fastest, most processive helicases known and contains a potent nuclease controlled by Chi sites, hotspots of recombination, in DNA. RecBCD undergoes major changes in activity and conformation when, during DNA unwinding, it encounters Chi (5'-GCTGGTGG-3') and nicks DNA nearby. Here, we discuss the multitude of mutations in each subunit that affect one or another activity of RecBCD and its control by Chi. These mutants have given deep insights into how the multiple activities of this complex enzyme are coordinated and how it acts in living cells. Similar studies could help reveal how other complex enzymes are controlled by inter-subunit interactions and conformational changes.}, }
@article {pmid37905078, year = {2023}, author = {Fazio, N and Mersch, KN and Hao, L and Lohman, TM}, title = {E. coli RecBCD Nuclease Domain Regulates Helicase Activity but not Single Stranded DNA Translocase Activity.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2023.10.13.561901}, pmid = {37905078}, abstract = {Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the E. coli RecBCD helicase can occur in the absence of ssDNA translocation of the canonical RecB and RecD motors. Here, we present evidence that dsDNA unwinding is not a simple consequence of ssDNA translocation by the RecBCD motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB [ΔNuc] CD) unwinds dsDNA at significantly slower rates than RecBCD, while the rate of ssDNA translocation is unaffected. This effect is primarily due to the absence of the nuclease domain and not the absence of the nuclease activity, since a nuclease-dead mutant (RecB [D1080A] CD), which retains the nuclease domain, showed no significant change in rates of ssDNA translocation or dsDNA unwinding relative to RecBCD on short DNA substrates (≤ 60 base pairs). This indicates that ssDNA translocation is not rate-limiting for DNA unwinding. RecB [ΔNuc] CD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecB [ΔNuc] CD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecB [D1080A] CD unwinding are intermediate between RecBCD and RecB [ΔNuc] CD. Surprisingly, significant pauses occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, rather than DNA translocation, possibly allosterically. Since the rate of DNA unwinding by RecBCD also slows after it recognizes a chi sequence, RecB [ΔNuc] CD may mimic a post- chi state of RecBCD.}, }
@article {pmid37104018, year = {2023}, author = {Fochtman, TJ and Oza, JP}, title = {Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems.}, journal = {Methods and protocols}, volume = {6}, number = {2}, pages = {}, doi = {10.3390/mps6020036}, pmid = {37104018}, issn = {2409-9279}, abstract = {Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro. However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design-build-test-learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field.}, }
@article {pmid37102866, year = {2023}, author = {Olina, A and Agapov, A and Yudin, D and Sutormin, D and Galivondzhyan, A and Kuzmenko, A and Severinov, K and Aravin, AA and Kulbachinskiy, A}, title = {Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0414622}, doi = {10.1128/spectrum.04146-22}, pmid = {37102866}, issn = {2165-0497}, abstract = {Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species. IMPORTANCE Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions in vivo. In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.}, }
@article {pmid36781123, year = {2023}, author = {Hamilton, NA and Jehru, AE and Samples, WN and Wendel, BM and Mokhtari, PD and Courcelle, CT and Courcelle, J}, title = {chi sequences switch the RecBCD helicase-nuclease complex from degradative to replicative modes during the completion of DNA replication.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {103013}, doi = {10.1016/j.jbc.2023.103013}, pmid = {36781123}, issn = {1083-351X}, abstract = {Accurately completing DNA replication when two forks converge is essential to genomic stability. The RecBCD helicase-nuclease complex plays a central role in completion by promoting resection and joining of the excess DNA created when replisomes converge. chi sequences alter RecBCD activity and localize with cross-over hotspots during sexual events in bacteria, yet their functional role during chromosome replication remains unknown. Here, we use two-dimensional agarose gel analysis to show that chi induces replication on substrates containing convergent forks. The induced-replication is processive, but uncoupled with respect to leading and lagging strand synthesis, and can be suppressed by ter sites which limit replisome progression. Our observations demonstrate that convergent replisomes create a substrate that is processed by RecBCD, and that chi, when encountered, switches RecBCD from a degradative to replicative function. We propose that chi serves to functionally differentiate DNA ends created during completion, which require degradation, from those created by chromosomal double-strand breaks, which require resynthesis.}, }
@article {pmid36533901, year = {2022}, author = {Wilkinson, M and Wilkinson, OJ and Feyerherm, C and Fletcher, EE and Wigley, DB and Dillingham, MS}, title = {Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub.}, journal = {eLife}, volume = {11}, number = {}, pages = {}, pmid = {36533901}, issn = {2050-084X}, support = {100401/Z/12/Z/WT_/Wellcome Trust/United Kingdom ; BB/S007261/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; C6913/A2160/CRUK_/Cancer Research UK/United Kingdom ; 209327/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; MR/N009258/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {*Escherichia coli Proteins/metabolism ; Escherichia coli/genetics/metabolism ; *Bacteriophages/genetics/metabolism ; Exodeoxyribonuclease V/genetics ; DNA/metabolism ; DNA, Single-Stranded/metabolism ; Recombinases/metabolism ; Exodeoxyribonucleases/chemistry/genetics/metabolism ; DNA, Bacterial/metabolism ; }, abstract = {Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.}, }
@article {pmid36521180, year = {2022}, author = {Amundsen, SK and Richardson, A and Ha, K and Smith, GR}, title = {A flexible RecC surface loop required for Chi hotspot control of RecBCD enzyme.}, journal = {Genetics}, volume = {}, number = {}, pages = {}, doi = {10.1093/genetics/iyac175}, pmid = {36521180}, issn = {1943-2631}, support = {R35 GM118120/GM/NIGMS NIH HHS/United States ; }, abstract = {Escherichia coli RecBCD helicase-nuclease promotes vital homologous recombination-based repair of DNA double-strand breaks. The RecB nuclease domain (Nuc) is connected to the RecB helicase domain by a 19-amino-acid tether. When DNA binds to RecBCD, published evidence suggests that Nuc moves ∼50 Å from the exit of a RecC tunnel, from which the 3'-ended strand emerges during unwinding, to a distant position on RecC's surface. During subsequent ATP-dependent unwinding of DNA, Nuc nicks the 3'-ended strand near 5' GCTGGTGG 3' (Chi recombination hotspot). Here, we test our model of Nuc swinging on the tether from the RecC tunnel exit to the RecC distant surface and back to the RecC tunnel exit to cut at Chi. We identify positions in a flexible surface loop on RecC and on RecB Nuc with complementary charges, mutation of which strongly reduces but does not eliminate Chi hotspot activity in cells. The recC loop mutation interacts with recB mutations hypothesized to be in the Chi-activated intramolecular signal transduction pathway; the double mutants, but not the single mutants, eliminate Chi hotspot activity. A RecC amino acid near the flexible loop is also essential for full Chi activity; its alteration likewise synergizes with a signal transduction mutation to eliminate Chi activity. We infer that altering the RecC surface loop reduces co-ordination among the subunits, which is critical for Chi hotspot activity. We discuss other RecBCD mutants with related properties.}, }
@article {pmid36334915, year = {2022}, author = {Subramaniam, S and Smith, GR}, title = {RecBCD enzyme and Chi recombination hotspots as determinants of self vs. non-self: Myths and mechanisms.}, journal = {Advances in genetics}, volume = {109}, number = {}, pages = {1-37}, doi = {10.1016/bs.adgen.2022.06.001}, pmid = {36334915}, issn = {0065-2660}, support = {R35 GM118120/GM/NIGMS NIH HHS/United States ; }, mesh = {Exodeoxyribonuclease V/genetics ; *Escherichia coli/genetics ; *Recombination, Genetic ; DNA Helicases/genetics ; DNA/genetics ; }, abstract = {Bacteria face a challenge when DNA enters their cells by transformation, mating, or phage infection. Should they treat this DNA as an invasive foreigner and destroy it, or consider it one of their own and potentially benefit from incorporating new genes or alleles to gain useful functions? It is frequently stated that the short nucleotide sequence Chi (5' GCTGGTGG 3'), a hotspot of homologous genetic recombination recognized by Escherichia coli's RecBCD helicase-nuclease, allows E. coli to distinguish its DNA (self) from any other DNA (non-self) and to destroy non-self DNA, and that Chi is "over-represented" in the E. coli genome. We show here that these latter statements (dogmas) are not supported by available evidence. We note Chi's wide-spread occurrence and activity in distantly related bacterial species and phages. We illustrate multiple, highly non-random features of the genomes of E. coli and coliphage P1 that account for Chi's high frequency and genomic position, leading us to propose that P1 selects for Chi's enhancement of recombination, whereas E. coli selects for the preferred codons in Chi. We discuss other, previously described mechanisms for self vs. non-self determination involving RecBCD and for RecBCD's destruction of DNA that cannot recombine, whether foreign or domestic, with or without Chi.}, }
@article {pmid36030574, year = {2022}, author = {Wang, BB and Xu, JZ and Zhang, F and Liu, S and Liu, J and Zhang, WG}, title = {Review of DNA repair enzymes in bacteria: With a major focus on AddAB and RecBCD.}, journal = {DNA repair}, volume = {118}, number = {}, pages = {103389}, doi = {10.1016/j.dnarep.2022.103389}, pmid = {36030574}, issn = {1568-7856}, mesh = {Bacillus subtilis ; DNA/metabolism ; DNA Repair ; DNA Repair Enzymes/metabolism ; DNA, Bacterial/metabolism ; *Escherichia coli/genetics ; Exodeoxyribonuclease V/metabolism ; *Exodeoxyribonucleases/metabolism ; }, abstract = {DNA recombination repair systems are essential for organisms to maintain genomic stability. In recent years, we have improved our understanding of the mechanisms of RecBCD/AddAB family-mediated DNA double-strand break repair. In E. coli, it is RecBCD that plays a central role, and in Firmicute Bacillus subtilis it is the AddAB complex that functions. However, there are open questions about the mechanism of DNA repair in bacteria. For example, how bacteria containing crossover hotspot instigator (Chi) sites regulate the activity of proteins. In addition, we still do not know the exact process by which the RecB nuclease or AddA nuclease structural domains load RecA onto DNA. We also know little about the mechanism of DNA repair in the industrially important production bacterium Corynebacterium glutamicum (C. glutamicum). Therefore, exploring DNA repair mechanisms in bacteria may not only deepen our understanding of the DNA repair process in this species but also guide us in the targeted treatment of diseases associated with recombination defects, such as cancer. In this paper, we firstly review the classical proteins RecBCD and AddAB involved in DNA recombination repair, secondly focus on the novel helical nuclease AdnAB found in the genus Mycobacterium.}, }
@article {pmid34432790, year = {2021}, author = {Yasmin, T and Azeroglu, B and Cockram, CA and Leach, DRF}, title = {Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair.}, journal = {PLoS genetics}, volume = {17}, number = {8}, pages = {e1009717}, pmid = {34432790}, issn = {1553-7404}, support = {MR/M019160/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Bacterial Proteins/genetics ; Chromosomes, Bacterial/metabolism ; DNA Breaks, Double-Stranded ; DNA Helicases/genetics ; DNA Repair/*genetics/physiology ; DNA Replication ; DNA, Bacterial/genetics ; DNA, Cruciform/*genetics/metabolism ; Escherichia coli/*genetics ; Escherichia coli Proteins/genetics ; Exodeoxyribonucleases/genetics/metabolism ; Homologous Recombination ; }, abstract = {Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.}, }
@article {pmid33561247, year = {2021}, author = {Buton, A and Bobay, LM}, title = {Evolution of Chi motifs in Proteobacteria.}, journal = {G3 (Bethesda, Md.)}, volume = {11}, number = {1}, pages = {}, pmid = {33561247}, issn = {2160-1836}, support = {R01 GM132137/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA, Bacterial ; *Escherichia coli/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics ; Proteobacteria ; *Recombination, Genetic ; }, abstract = {Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex not only allows bacteria to repair DNA double-strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis, and Staphylococcus aureus. In this study, we detected putative Chi motifs in a large dataset of Proteobacteria and identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.}, }
@article {pmid33154402, year = {2020}, author = {Amundsen, SK and Taylor, AF and Smith, GR}, title = {Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {19415}, pmid = {33154402}, issn = {2045-2322}, support = {P30 CA015704/CA/NCI NIH HHS/United States ; R35 GM118120/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; Binding Sites ; DNA Helicases/genetics/*metabolism ; DNA Repair/genetics ; DNA, Bacterial/genetics/metabolism ; Endonucleases/genetics/*metabolism ; Escherichia coli/*enzymology/genetics ; Escherichia coli Proteins/*genetics/*metabolism ; Exodeoxyribonuclease V/*genetics/*metabolism ; Models, Molecular ; Mutation ; Protein Structure, Quaternary ; *Recombination, Genetic ; Signal Transduction ; }, abstract = {Repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5' GCTGGTGG 3') recognized during DNA unwinding. Chi is recognized in a tunnel in RecC but activates the RecB nuclease, > 25 Ǻ away. How the Chi-dependent signal travels this long distance has been unknown. We found a Chi hotspot-deficient mutant in the RecB helicase domain located > 45 Ǻ from both the Chi-recognition site and the nuclease active site. This unexpected observation led us to find additional mutations that reduced or eliminated Chi hotspot activity in each subunit and widely scattered throughout RecBCD. Each mutation alters the intimate contact between one or another pair of subunits in crystal or cryoEM structures of RecBCD bound to DNA. Collectively, these mutations span a path about 185 Ǻ long from the Chi recognition site to the nuclease active site. We discuss these surprising results in the context of an intramolecular signal transduction accounting for many previous observations.}, }
@article {pmid33085588, year = {2020}, author = {Gurung, D and Blumenthal, RM}, title = {Distribution of RecBCD and AddAB recombination-associated genes among bacteria in 33 phyla.}, journal = {Microbiology (Reading, England)}, volume = {166}, number = {11}, pages = {1047-1064}, doi = {10.1099/mic.0.000980}, pmid = {33085588}, issn = {1465-2080}, mesh = {Amino Acid Sequence ; Bacteria/classification/*genetics ; Base Sequence ; Conserved Sequence ; DNA, Bacterial/genetics ; Evolution, Molecular ; Exodeoxyribonuclease V/*genetics ; Exodeoxyribonucleases/*genetics ; Gene Transfer, Horizontal/genetics ; Phylogeny ; Rec A Recombinases/genetics ; Recombination, Genetic/*genetics ; }, abstract = {Homologous recombination plays key roles in fundamental processes such as recovery from DNA damage and in bacterial horizontal gene transfer, yet there are still open questions about the distribution of recognized components of recombination machinery among bacteria and archaea. RecBCD helicase-nuclease plays a central role in recombination among Gammaproteobacteria like Escherichia coli; while bacteria in other phyla, like the Firmicute Bacillus subtilis, use the related AddAB complex. The activity of at least some of these complexes is controlled by short DNA sequences called crossover hotspot instigator (Chi) sites. When RecBCD or AddAB complexes encounter an autologous Chi site during unwinding, they introduce a nick such that ssDNA with a free end is available to invade another duplex. If homologous DNA is present, RecA-dependent homologous recombination is promoted; if not (or if no autologous Chi site is present) the RecBCD/AddAB complex eventually degrades the DNA. We examined the distribution of recBCD and addAB genes among bacteria, and sought ways to distinguish them unambiguously. We examined bacterial species among 33 phyla, finding some unexpected distribution patterns. RecBCD and addAB are less conserved than recA, with the orthologous recB and addA genes more conserved than the recC or addB genes. We were able to classify RecB vs. AddA and RecC vs. AddB in some bacteria where this had not previously been done. We used logo analysis to identify sequence segments that are conserved, but differ between the RecBC and AddAB proteins, to help future differentiation between members of these two families.}, }
@article {pmid32926785, year = {2020}, author = {Yim, SS and Johns, NI and Noireaux, V and Wang, HH}, title = {Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria.}, journal = {ACS synthetic biology}, volume = {9}, number = {10}, pages = {2851-2855}, doi = {10.1021/acssynbio.0c00277}, pmid = {32926785}, issn = {2161-5063}, support = {U01 GM110714/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*metabolism ; Base Sequence ; Cell-Free System ; DNA, Bacterial/*metabolism ; Enzyme Inhibitors/pharmacology ; Escherichia coli/*enzymology/*genetics ; Escherichia coli Proteins/antagonists & inhibitors/*metabolism ; Exodeoxyribonuclease V/antagonists & inhibitors/*metabolism ; Exonucleases/antagonists & inhibitors/*metabolism ; Genes, Bacterial ; Mycobacterium tuberculosis/*genetics ; Plasmids/genetics ; Recombinant Proteins/metabolism ; Synthetic Biology/methods ; Transcription, Genetic ; Viral Proteins/metabolism ; }, abstract = {Recent advances in cell-free systems have opened up new capabilities in synthetic biology from rapid prototyping of genetic circuits and metabolic pathways to portable diagnostics and biomanufacturing. A current bottleneck in cell-free systems, especially those employing non-E. coli bacterial species, is the required use of plasmid DNA, which can be laborious to construct, clone, and verify. Linear DNA templates offer a faster and more direct route for many cell-free applications, but they are often rapidly degraded in cell-free reactions. In this study, we evaluated GamS from λ-phage, DNA fragments containing Chi-sites, and Ku from Mycobacterium tuberculosis for their ability to protect linear DNA templates in diverse bacterial cell-free systems. We show that these nuclease inhibitors exhibit differential protective activities against endogenous exonucleases in five different cell-free lysates, highlighting their utility for diverse bacterial species. We expect these linear DNA protection strategies will accelerate high-throughput approaches in cell-free synthetic biology.}, }
@article {pmid32597964, year = {2020}, author = {Karabulut, AC and Cirz, RT and Taylor, AF and Smith, GR}, title = {Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state.}, journal = {Nucleic acids research}, volume = {48}, number = {14}, pages = {7973-7980}, pmid = {32597964}, issn = {1362-4962}, support = {R35 GM118120/GM/NIGMS NIH HHS/United States ; R01 GM031693/GM/NIGMS NIH HHS/United States ; P30 CA015704/CA/NCI NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/metabolism ; Benzimidazoles/chemistry/*pharmacology ; Binding Sites ; Enzyme Inhibitors/chemistry/*pharmacology ; Exodeoxyribonuclease V/*antagonists & inhibitors/chemistry/genetics/metabolism ; Mutation ; }, abstract = {Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.}, }
@article {pmid31907455, year = {2020}, author = {Cheng, K and Wilkinson, M and Chaban, Y and Wigley, DB}, title = {A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair.}, journal = {Nature structural & molecular biology}, volume = {27}, number = {1}, pages = {71-77}, pmid = {31907455}, issn = {1545-9985}, support = {/WT_/Wellcome Trust/United Kingdom ; 12799/CRUK_/Cancer Research UK/United Kingdom ; A12799/CRUK_/Cancer Research UK/United Kingdom ; MR/N009258/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Bacteriophage lambda/physiology ; Base Sequence ; Binding Sites ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cryoelectron Microscopy ; *DNA Repair ; DNA, Bacterial/chemistry/*metabolism/ultrastructure ; Escherichia coli/chemistry/*metabolism ; Escherichia coli Proteins/chemistry/*metabolism/ultrastructure ; Exodeoxyribonuclease V/chemistry/*metabolism/ultrastructure ; Molecular Docking Simulation ; Protein Conformation ; }, abstract = {The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.}, }
@article {pmid31719908, year = {2019}, author = {Xie, P}, title = {Modeling DNA Unwinding by AddAB Helicase-Nuclease and Modulation by Chi Sequences: Comparison with AdnAB and RecBCD.}, journal = {Cellular and molecular bioengineering}, volume = {12}, number = {2}, pages = {179-191}, pmid = {31719908}, issn = {1865-5025}, abstract = {INTRODUCTION: AddAB enzyme is a helicase-nuclease complex that initiates recombinational repair of double-stranded DNA breaks. It catalyzes processive DNA unwinding and concomitant resection of the unwound strands, which are modulated by the recognition of a recombination hotspot called Chi in the 3'-terminated strand. Despite extensive structural, biochemical and single molecule studies, the detailed molecular mechanism of DNA unwinding by the complex and modulation by Chi sequence remains unclear.<br><br>METHODS: A model of DNA unwinding by the AddAB complex and modulation by Chi recognition was presented, based on which the dynamics of AddAB complex was studied analytically.<br><br>RESULTS: The theoretical results explain well the available experimental data on effect of DNA sequence on velocity, effect of Chi recognition on velocity, static disorder peculiar to the AddAB complex, and dynamics of pausing of wild-type and mutant AddAB complexes occurring at Chi or Chi-like sequence. Predictions were provided. Comparisons of AddAB complex with other helicase-nuclease complexes such as RecBCD and AdnAB were made.<br><br>CONCLUSIONS: The study has strong implications for the molecular mechanism of DNA unwinding by the AddAB complex. The intriguing issues are addressed of why Chi recognition is an inefficient process, how AddAB complex pauses upon recognizing Chi sequence, how the paused state transits to the translocating state, why the mutant AddAB with a stronger affinity to Chi sequence has a shorter pausing lifetime, why the pausing lifetime is sensitive to the solution temperature, and so on.}, }
@article {pmid30544167, year = {2019}, author = {Li, C and Danilowicz, C and Tashjian, TF and Godoy, VG and Prévost, C and Prentiss, M}, title = {The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements.}, journal = {Nucleic acids research}, volume = {47}, number = {4}, pages = {1836-1846}, pmid = {30544167}, issn = {1362-4962}, mesh = {Base Sequence ; DNA/biosynthesis/*genetics ; DNA Breaks, Double-Stranded ; DNA Repair/genetics ; DNA Replication/genetics ; DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; Exodeoxyribonuclease V/*genetics ; Genome, Bacterial/*genetics ; Nucleic Acid Heteroduplexes/genetics ; Rec A Recombinases/genetics ; Recombination, Genetic ; }, abstract = {Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3' single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3' ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3' end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.}, }
@article {pmid30445486, year = {2019}, author = {Amundsen, SK and Smith, GR}, title = {The RecB helicase-nuclease tether mediates Chi hotspot control of RecBCD enzyme.}, journal = {Nucleic acids research}, volume = {47}, number = {1}, pages = {197-209}, pmid = {30445486}, issn = {1362-4962}, support = {P30 CA015704/CA/NCI NIH HHS/United States ; R01 GM031693/GM/NIGMS NIH HHS/United States ; R35 GM118120/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Sequence/genetics ; Cell-Free System ; DNA Breaks, Double-Stranded ; DNA Helicases/*genetics ; DNA Repair/genetics ; Escherichia coli/genetics ; Escherichia coli Proteins/*genetics ; Exodeoxyribonuclease V/*genetics ; Gene Expression Regulation, Bacterial ; Glycine/genetics ; Nucleotide Motifs/genetics ; *Recombination, Genetic ; }, abstract = {In bacteria, repair of DNA double-strand breaks uses a highly conserved helicase-nuclease complex to unwind DNA from a broken end and cut it at specific DNA sequences called Chi. In Escherichia coli the RecBCD enzyme also loads the DNA strand-exchange protein RecA onto the newly formed end, resulting in a recombination hotspot at Chi. Chi hotspots regulate multiple RecBCD activities by altering RecBCD's conformation, which is proposed to include the swinging of the RecB nuclease domain on the 19-amino-acid tether connecting the helicase and nuclease domains. Here, we altered the tether and tested multiple RecBCD activities, genetically in cells and enzymatically in cell-free extracts. Randomizing the amino-acid sequence or lengthening it had little effect. However, shortening it by as little as two residues or making substitutions of ≥10 proline or ≥9 glycine residues dramatically lowered Chi-dependent activities. These results indicate that proper control of RecBCD by Chi requires that the tether be long enough and appropriately flexible. We discuss a model in which the swing-time of the nuclease domain determines the position of Chi-dependent and Chi-independent cuts and Chi hotspot activity.}, }
@article {pmid29775464, year = {2018}, author = {Pavankumar, TL and Sinha, AK and Ray, MK}, title = {Biochemical characterization of RecBCD enzyme from an Antarctic Pseudomonas species and identification of its cognate Chi (χ) sequence.}, journal = {PloS one}, volume = {13}, number = {5}, pages = {e0197476}, pmid = {29775464}, issn = {1932-6203}, mesh = {Adenosine Triphosphatases/metabolism ; Adenosine Triphosphate/metabolism/pharmacology ; Antarctic Regions ; Base Sequence ; Cloning, Molecular ; DNA/metabolism ; DNA, Bacterial/metabolism ; Exodeoxyribonuclease V/isolation &amp; purification/*metabolism ; Hydrolysis ; Magnesium/pharmacology ; Mutant Proteins/metabolism ; Mutation/genetics ; Plasmids/metabolism ; Pseudomonas/*enzymology ; Pseudomonas syringae/enzymology ; Recombinant Fusion Proteins/isolation &amp; purification ; Substrate Specificity/drug effects ; Temperature ; }, abstract = {Pseudomonas syringae Lz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential for P. syringae growth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPs enzyme displays distinct biochemical behaviors. Unlike E. coli RecBCD enzyme, the RecD subunit is indispensable for RecBCDPs function. The RecD motor activity is essential for the Chi-like fragments production in P. syringae, highlighting a distinct role for P. syringae RecD subunit in DNA repair and recombination process. Here, we demonstrate that the RecBCDPs enzyme recognizes a unique octameric DNA sequence, 5'-GCTGGCGC-3' (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 3'-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity in P. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest.}, }
@article {pmid29655868, year = {2018}, author = {Xie, P}, title = {A model of DNA unwinding dynamics by the RecBCD complex and its regulation by Chi recognition.}, journal = {Journal of theoretical biology}, volume = {448}, number = {}, pages = {142-156}, doi = {10.1016/j.jtbi.2018.04.014}, pmid = {29655868}, issn = {1095-8541}, mesh = {Base Sequence ; DNA Breaks, Double-Stranded ; DNA Repair/*physiology ; DNA, Bacterial/genetics ; DNA, Single-Stranded/genetics ; Escherichia coli/*genetics ; Escherichia coli Proteins/*genetics/physiology ; Exodeoxyribonuclease V/metabolism ; *Models, Molecular ; }, abstract = {The Escherichia coli RecBCD enzyme is a heterotrimeric helicase-nuclease complex responsible for processing of double-stranded DNA breaks for repair by homologous recombination. It is a highly processive, duplex unwinding and degrading motor, with its activities being regulated by the octameric recombination hotspot, Chi, which is read as a single-stranded DNA sequence. Here, a model is presented for DNA unwinding by the RecBCD complex and its regulation by Chi recognition. With the model we study analytically the dynamics of DNA unwinding of both wild-type RecBCD and mutant RecBCD[K177Q] with the motor function of RecD being inactivated by mutagenesis, giving quantitative explanations of the available single-molecule experimental data. The peculiar features of RecBCD such as large variations of DNA unwinding speed of individual enzymes, sensitivity of unwinding speed of a RecBCD molecule on the change of environment, two translocase or helicase activities of RecBC and RecD, etc., are explained. Furthermore, predicted results are presented.}, }
@article {pmid32995510, year = {2018}, author = {Klocke, MA and Garamella, J and Subramanian, HKK and Noireaux, V and Franco, E}, title = {Engineering DNA nanotubes for resilience in an E. coli TXTL system.}, journal = {Synthetic biology (Oxford, England)}, volume = {3}, number = {1}, pages = {ysy001}, pmid = {32995510}, issn = {2397-7000}, abstract = {Deoxyribonucleic acid (DNA) nanotechnology is a growing field with potential intracellular applications. In this work, we use an Escherichia coli cell-free transcription-translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modifications of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 h, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site double-stranded DNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime significantly. These complementary approaches are a first step toward a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering.}, }
@article {pmid29032606, year = {2018}, author = {Cho, CC and Chung, C and Li, HW}, title = {How Chi Sequence Modifies RecBCD Single-Stranded DNA Translocase Activity.}, journal = {Chemphyschem : a European journal of chemical physics and physical chemistry}, volume = {19}, number = {2}, pages = {243-247}, doi = {10.1002/cphc.201700840}, pmid = {29032606}, issn = {1439-7641}, mesh = {Biological Transport ; DNA, Single-Stranded/*genetics/*metabolism ; Escherichia coli/enzymology ; Escherichia coli Proteins/*metabolism ; Exodeoxyribonuclease V/*metabolism ; }, abstract = {E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCD[K177Q] showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.}, }
@article {pmid28976965, year = {2017}, author = {Sivaramakrishnan, P and Sepúlveda, LA and Halliday, JA and Liu, J and Núñez, MAB and Golding, I and Rosenberg, SM and Herman, C}, title = {The transcription fidelity factor GreA impedes DNA break repair.}, journal = {Nature}, volume = {550}, number = {7675}, pages = {214-218}, pmid = {28976965}, issn = {1476-4687}, support = {R01 GM082837/GM/NIGMS NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; R01 GM088653/GM/NIGMS NIH HHS/United States ; R35 GM122598/GM/NIGMS NIH HHS/United States ; DP1 CA174424/CA/NCI NIH HHS/United States ; }, mesh = {DNA Breaks, Double-Stranded ; *DNA Repair ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/enzymology/*genetics/*metabolism ; Escherichia coli Proteins/*metabolism ; Exodeoxyribonuclease V/metabolism ; Protein Binding ; Rec A Recombinases/metabolism ; Transcription Factors/*metabolism ; *Transcription, Genetic ; }, abstract = {Homologous recombination repairs DNA double-strand breaks and must function even on actively transcribed DNA. Because break repair prevents chromosome loss, the completion of repair is expected to outweigh the transcription of broken templates. However, the interplay between DNA break repair and transcription processivity is unclear. Here we show that the transcription factor GreA inhibits break repair in Escherichia coli. GreA restarts backtracked RNA polymerase and hence promotes transcription fidelity. We report that removal of GreA results in markedly enhanced break repair via the classic RecBCD-RecA pathway. Using a deep-sequencing method to measure chromosomal exonucleolytic degradation, we demonstrate that the absence of GreA limits RecBCD-mediated resection. Our findings suggest that increased RNA polymerase backtracking promotes break repair by instigating RecA loading by RecBCD, without the influence of canonical Chi signals. The idea that backtracked RNA polymerase can stimulate recombination presents a DNA transaction conundrum: a transcription fidelity factor that compromises genomic integrity.}, }
@article {pmid28968392, year = {2017}, author = {Sinha, AK and Durand, A and Desfontaines, JM and Iurchenko, I and Auger, H and Leach, DRF and Barre, FX and Michel, B}, title = {Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme.}, journal = {PLoS genetics}, volume = {13}, number = {10}, pages = {e1006895}, pmid = {28968392}, issn = {1553-7404}, support = {MR/K001744/1/MRC_/Medical Research Council/United Kingdom ; MR/M019160/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Cell Division ; Chromosomes, Bacterial/*genetics ; *DNA Breaks, Double-Stranded ; DNA Repair ; DNA Replication ; DNA, Bacterial/genetics ; Escherichia coli/*genetics ; Escherichia coli Proteins/*genetics/metabolism ; Exodeoxyribonuclease V/*genetics/metabolism ; Sequence Analysis, DNA ; }, abstract = {Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.}, }
@article {pmid28475211, year = {2017}, author = {Marshall, R and Maxwell, CS and Collins, SP and Beisel, CL and Noireaux, V}, title = {Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems.}, journal = {Biotechnology and bioengineering}, volume = {114}, number = {9}, pages = {2137-2141}, pmid = {28475211}, issn = {1097-0290}, support = {R35 GM119561/GM/NIGMS NIH HHS/United States ; }, mesh = {Cell-Free System/physiology ; DNA, Bacterial/*genetics ; Escherichia coli/*genetics ; Exodeoxyribonuclease V/*genetics ; Gene Expression Regulation, Bacterial/*genetics ; Genetic Engineering/*methods ; Protein Biosynthesis/*genetics ; Transcription, Genetic/*genetics ; }, abstract = {Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137-2141. © 2017 Wiley Periodicals, Inc.}, }
@article {pmid27401752, year = {2016}, author = {Amundsen, SK and Sharp, JW and Smith, GR}, title = {RecBCD Enzyme "Chi Recognition" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context.}, journal = {Genetics}, volume = {204}, number = {1}, pages = {139-152}, pmid = {27401752}, issn = {1943-2631}, support = {R01 GM031693/GM/NIGMS NIH HHS/United States ; R56 GM031693/GM/NIGMS NIH HHS/United States ; }, mesh = {Base Sequence ; Binding Sites ; DNA/*genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA Helicases/genetics/metabolism ; DNA Repair ; DNA, Bacterial/genetics/metabolism ; Endonucleases/metabolism ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Exodeoxyribonuclease V/chemistry/*genetics/metabolism ; Models, Molecular ; Rec A Recombinases/genetics ; Recombination, Genetic ; }, abstract = {RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.}, }
@article {pmid27330137, year = {2016}, author = {Taylor, AF and Amundsen, SK and Smith, GR}, title = {Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots.}, journal = {Nucleic acids research}, volume = {44}, number = {17}, pages = {8216-8228}, pmid = {27330137}, issn = {1362-4962}, support = {R01 GM031693/GM/NIGMS NIH HHS/United States ; R56 GM031693/GM/NIGMS NIH HHS/United States ; }, mesh = {Base Sequence ; DNA/*genetics ; Escherichia coli/drug effects/genetics ; Escherichia coli Proteins/metabolism ; Exodeoxyribonuclease V/metabolism ; Ions ; Magnesium/pharmacology ; Models, Genetic ; Nucleotides/genetics ; *Recombination, Genetic ; }, abstract = {Homologous recombination occurs especially frequently near special chromosomal sites called hotspots. In Escherichia coli, Chi hotspots control RecBCD enzyme, a protein machine essential for the major pathway of DNA break-repair and recombination. RecBCD generates recombinogenic single-stranded DNA ends by unwinding DNA and cutting it a few nucleotides to the 3' side of 5' GCTGGTGG 3', the sequence historically equated with Chi. To test if sequence context affects Chi activity, we deep-sequenced the products of a DNA library containing 10 random base-pairs on each side of the Chi sequence and cut by purified RecBCD. We found strongly enhanced cutting at Chi with certain preferred sequences, such as A or G at nucleotides 4-7, on the 3' flank of the Chi octamer. These sequences also strongly increased Chi hotspot activity in E. coli cells. Our combined enzymatic and genetic results redefine the Chi hotspot sequence, implicate the nuclease domain in Chi recognition, indicate that nicking of one strand at Chi is RecBCD's biologically important reaction in living cells, and enable more precise analysis of Chi's role in recombination and genome evolution.}, }
@article {pmid26261330, year = {2015}, author = {Cockram, CA and Filatenkova, M and Danos, V and El Karoui, M and Leach, DR}, title = {Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, number = {34}, pages = {E4735-42}, pmid = {26261330}, issn = {1091-6490}, support = {G0901622/MRC_/Medical Research Council/United Kingdom ; MR/M019160/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Chromatin Immunoprecipitation ; *DNA Damage ; *DNA Repair ; Exodeoxyribonuclease V/*metabolism ; *Genome ; Rec A Recombinases/genetics/*metabolism ; }, abstract = {Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.}, }
@article {pmid25874675, year = {2015}, author = {Levy, A and Goren, MG and Yosef, I and Auster, O and Manor, M and Amitai, G and Edgar, R and Qimron, U and Sorek, R}, title = {CRISPR adaptation biases explain preference for acquisition of foreign DNA.}, journal = {Nature}, volume = {520}, number = {7548}, pages = {505-510}, pmid = {25874675}, issn = {1476-4687}, support = {260432/ERC_/European Research Council/International ; 336079/ERC_/European Research Council/International ; }, mesh = {*Adaptation, Physiological ; Bacteriophages/*genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Consensus Sequence/genetics ; DNA Breaks, Double-Stranded ; DNA Repair ; DNA Replication/genetics ; DNA, Bacterial/*genetics ; DNA, Viral/*genetics ; Escherichia coli/*genetics ; Exodeoxyribonuclease V/metabolism ; Models, Biological ; Plasmids/*genetics ; }, abstract = {CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.}, }
@article {pmid25486468, year = {2014}, author = {Wilkinson, M and Wigley, DB}, title = {Structural features of Chi recognition in AddAB with implications for RecBCD.}, journal = {Cell cycle (Georgetown, Tex.)}, volume = {13}, number = {18}, pages = {2812-2820}, pmid = {25486468}, issn = {1551-4005}, support = {12799/CRUK_/Cancer Research UK/United Kingdom ; }, mesh = {Bacillus subtilis/*enzymology ; *Crossing Over, Genetic ; Escherichia coli/*enzymology ; Exodeoxyribonuclease V/*chemistry/*metabolism ; Exodeoxyribonucleases/*chemistry/*metabolism ; Models, Molecular ; Protein Binding ; Protein Structure, Tertiary ; Structural Homology, Protein ; }, abstract = {AddAB and RecBCD-type helicase-nuclease complexes control the first stage of bacterial homologous recombination (HR) - the resection of double strand DNA breaks. A switch in the activities of the complexes to initiate repair by HR is regulated by a short, species-specific DNA sequence known as a Crossover Hotspot Instigator (Chi) site. It has been shown that, upon encountering Chi, AddAB and RecBCD pause translocation before resuming at a reduced rate. Recently, the structure of B.subtilis AddAB in complex with its regulatory Chi sequence revealed the nature of Chi binding and the paused translocation state. Here the structural features associated with Chi binding are described in greater detail and discussed in relation to the related E.coli RecBCD system.}, }
@article {pmid25073102, year = {2014}, author = {Taylor, AF and Amundsen, SK and Guttman, M and Lee, KK and Luo, J and Ranish, J and Smith, GR}, title = {Control of RecBCD enzyme activity by DNA binding- and Chi hotspot-dependent conformational changes.}, journal = {Journal of molecular biology}, volume = {426}, number = {21}, pages = {3479-3499}, pmid = {25073102}, issn = {1089-8638}, support = {R00 GM080352/GM/NIGMS NIH HHS/United States ; R01 GM099989/GM/NIGMS NIH HHS/United States ; P41 RR001209/RR/NCRR NIH HHS/United States ; R01 GM031693/GM/NIGMS NIH HHS/United States ; P41-RR001209/RR/NCRR NIH HHS/United States ; P50 GM076547/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; Crystallography, X-Ray ; DNA Repair ; DNA, Single-Stranded/chemistry ; Escherichia coli/*metabolism ; Escherichia coli Proteins/*physiology ; Exodeoxyribonuclease V/*physiology ; *Gene Expression Regulation, Bacterial ; Magnesium/chemistry ; Mass Spectrometry ; Molecular Sequence Data ; Peptide Hydrolases/chemistry ; Protein Binding ; Protein Structure, Tertiary ; Recombination, Genetic ; Scattering, Radiation ; Trypsin/chemistry ; X-Rays ; }, abstract = {Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5' GCTGGTGG 3') during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3'-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular "signal transduction" from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.}, }
@article {pmid24670664, year = {2014}, author = {Krajewski, WW and Fu, X and Wilkinson, M and Cronin, NB and Dillingham, MS and Wigley, DB}, title = {Structural basis for translocation by AddAB helicase-nuclease and its arrest at χ sites.}, journal = {Nature}, volume = {508}, number = {7496}, pages = {416-419}, pmid = {24670664}, issn = {1476-4687}, support = {100401//Wellcome Trust/United Kingdom ; 12799/CRUK_/Cancer Research UK/United Kingdom ; A12799/CRUK_/Cancer Research UK/United Kingdom ; }, mesh = {Adenosine Triphosphate/analogs &amp; derivatives/metabolism ; Bacillus subtilis/*enzymology ; Bacterial Proteins/*chemistry/*metabolism ; Binding Sites ; Crystallography, X-Ray ; DNA/chemistry/genetics/metabolism ; DNA Helicases/*chemistry/metabolism ; Exodeoxyribonucleases/*chemistry/*metabolism ; Models, Molecular ; Molecular Conformation ; Recombination, Genetic/*genetics ; Structure-Activity Relationship ; }, abstract = {In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi) and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence, whereupon they produce a 3' single-stranded DNA tail onto which they initiate loading of the RecA protein. Consequently, regulation of the AddAB/RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments.}, }
@article {pmid24214339, year = {2014}, author = {Reams, AB and Kofoid, E and Duleba, N and Roth, JR}, title = {Recombination and annealing pathways compete for substrates in making rrn duplications in Salmonella enterica.}, journal = {Genetics}, volume = {196}, number = {1}, pages = {119-135}, pmid = {24214339}, issn = {1943-2631}, support = {R01 GM027068/GM/NIGMS NIH HHS/United States ; GM27068/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacterial Proteins/genetics ; Chromosomes, Bacterial/*genetics ; DNA Breaks, Double-Stranded ; DNA Helicases/genetics ; DNA Mismatch Repair/genetics ; DNA, Bacterial/*genetics ; DNA-Binding Proteins/genetics ; Exodeoxyribonuclease V/genetics ; RNA, Ribosomal/*genetics ; Rec A Recombinases/genetics ; Salmonella enterica/*genetics ; Tandem Repeat Sequences/*genetics ; rRNA Operon/genetics ; }, abstract = {Tandem genetic duplications arise frequently between the seven directly repeated 5.5-kb rrn loci that encode ribosomal RNAs in Salmonella enterica. The closest rrn genes, rrnB and rrnE, flank a 40-kb region that includes the purHD operon. Duplications of purHD arise by exchanges between rrn loci and form at a high rate (10(-3)/cell/division) that remains high in strains blocked for early steps in recombination (recA, recB, and/or recF), but drops 30-fold in mutants blocked for later Holliday junction resolution (ruvC recG). The duplication defect of a ruvC recG mutant was fully corrected by an added mutation in any one of the recA, recB, or recF genes. To explain these results, we propose that early recombination defects activate an alternative single-strand annealing pathway for duplication formation. In wild-type cells, rrn duplications form primarily by the action of RecFORA on single-strand gaps. Double-strand breaks cannot initiate rrn duplications because rrn loci lack Chi sites, which are essential for recombination between two separated rrn sequences. A recA or recF mutation allows unrepaired gaps to accumulate such that different rrn loci can provide single-strand rrn sequences that lack the RecA coating that normally inhibits annealing. A recB mutation activates annealing by allowing double-strand ends within rrn to avoid digestion by RecBCD and provide a new source of rrn ends for use in annealing. The equivalent high rates of rrn duplication by recombination and annealing pathways may reflect a limiting economy of gaps and breaks arising in heavily transcribed, palindrome-rich rrn sequences.}, }
@article {pmid24086157, year = {2013}, author = {Bobay, LM and Touchon, M and Rocha, EP}, title = {Manipulating or superseding host recombination functions: a dilemma that shapes phage evolvability.}, journal = {PLoS genetics}, volume = {9}, number = {9}, pages = {e1003825}, pmid = {24086157}, issn = {1553-7404}, mesh = {Bacteriophage lambda/*genetics ; DNA Replication/genetics ; Escherichia coli/*genetics ; Evolution, Molecular ; Exodeoxyribonuclease V/genetics ; Host-Pathogen Interactions/*genetics ; Nucleotide Motifs/genetics ; Recombinases/genetics ; *Recombination, Genetic ; }, abstract = {Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts'. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi) motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host's machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability.}, }
@article {pmid22688812, year = {2012}, author = {Smith, GR}, title = {How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist's view.}, journal = {Microbiology and molecular biology reviews : MMBR}, volume = {76}, number = {2}, pages = {217-228}, pmid = {22688812}, issn = {1098-5557}, support = {R01 GM031693/GM/NIGMS NIH HHS/United States ; R56 GM031693/GM/NIGMS NIH HHS/United States ; GM031693/GM/NIGMS NIH HHS/United States ; }, mesh = {*DNA Breaks, Double-Stranded ; *DNA Repair ; DNA, Bacterial/*metabolism ; Escherichia coli/*enzymology/genetics/metabolism ; Exodeoxyribonuclease V/chemistry/*metabolism ; Nuclear Proteins/chemistry/*metabolism ; }, abstract = {The repair of DNA double-strand breaks (DSBs) is essential for cell viability and important for homologous genetic recombination. In enteric bacteria such as Escherichia coli, the major pathway of DSB repair requires the RecBCD enzyme, a complex helicase-nuclease regulated by a simple unique DNA sequence called Chi. How Chi regulates RecBCD has been extensively studied by both genetics and biochemistry, and two contrasting mechanisms to generate a recombinogenic single-stranded DNA tail have been proposed: the nicking of one DNA strand at Chi versus the switching of degradation from one strand to the other at Chi. Which of these reactions occurs in cells has remained unproven because of the inability to detect intracellular DNA intermediates in bacterial recombination and DNA break repair. Here, I discuss evidence from a combination of genetics and biochemistry indicating that nicking at Chi is the intracellular (in vivo) reaction. This example illustrates the need for both types of analysis (i.e., molecular biology) to uncover the mechanism and control of complex processes in living cells.}, }
@article {pmid22617484, year = {2012}, author = {Vlašić, I and Simatović, A and Brčić-Kostić, K}, title = {The hybrid recombinational repair pathway operates in a χ activity deficient recC1004 mutant of Escherichia coli.}, journal = {Biochimie}, volume = {94}, number = {9}, pages = {1918-1925}, doi = {10.1016/j.biochi.2012.05.008}, pmid = {22617484}, issn = {1638-6183}, mesh = {DNA Breaks, Double-Stranded/radiation effects ; DNA Helicases/metabolism ; DNA Repair/*genetics/radiation effects ; Deoxyribonucleases/metabolism ; Escherichia coli/enzymology/*genetics/*metabolism ; Escherichia coli Proteins/*genetics/*metabolism ; Exodeoxyribonuclease V/*genetics/*metabolism ; *Mutation ; Recombination, Genetic/*genetics/radiation effects ; }, abstract = {Homologous recombination is a crucial process for the maintenance of genome integrity. The two main recombination pathways in Escherichia coli (RecBCD and RecF) differ in the initiation of recombination. The RecBCD enzyme is the only component of the RecBCD pathway which acts in the initiation of recombination, and possesses all biochemical activities (helicase, 5'-3' exonuclease, χ cutting and loading of the RecA protein onto single-stranded (ss) DNA) needed for the processing of double stranded (ds) DNA breaks (DSB). When the nuclease and RecA loading activities of the RecBCD enzyme are inactivated, the proteins of the RecF recombination machinery, i.e., RecJ and RecFOR substitute for the missing 5'-3' exonuclease and RecA loading activity respectively. The above mentioned activities of the RecBCD enzyme are regulated by an octameric sequence known as the χ site (5'-GCTGGTGG-3'). One class of recC mutations, designated recC*, leads to reduced χ cutting in vitro. The recC1004 strain (a member of the recC* mutant class) is recombination proficient and resistant to UV radiation. In this paper, we studied the effects of mutations in RecF pathway genes on DNA repair (after UV and γ radiation) and on conjugational recombination in recC1004 and recC1004 recD backgrounds. We found that DNA repair after UV and γ radiation in the recC1004 and recC1004 recD backgrounds depends on recFOR and recJ gene products. We also showed that the recC1004 mutant has reduced survival after γ radiation. This phenotype is suppressed by the recD mutation which abolishes the RecBCD dependent nuclease activity. Finally, the genetic requirements for conjugational recombination differ from those for DNA repair. Conjugational recombination in recC1004 recD mutants is dependent on the recJ gene product. Our results emphasize the importance of the canonical χ recognition activity in DSB repair and the significance of interchange between the components of two recombination machineries in achieving efficient DNA repair.}, }
@article {pmid22603794, year = {2012}, author = {Handa, N and Yang, L and Dillingham, MS and Kobayashi, I and Wigley, DB and Kowalczykowski, SC}, title = {Molecular determinants responsible for recognition of the single-stranded DNA regulatory sequence, χ, by RecBCD enzyme.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, number = {23}, pages = {8901-8906}, pmid = {22603794}, issn = {1091-6490}, support = {12799/CRUK_/Cancer Research UK/United Kingdom ; R01 GM041347/GM/NIGMS NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; GM41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; Computational Biology ; DNA Primers/genetics ; DNA Repair/*genetics/physiology ; DNA, Single-Stranded/genetics/*metabolism ; Escherichia coli/*enzymology ; Escherichia coli Proteins/genetics ; Exodeoxyribonuclease V/genetics/*metabolism ; *Models, Molecular ; Molecular Sequence Data ; Mutagenesis ; Plasmids/genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; Sequence Alignment ; }, abstract = {The RecBCD enzyme is important for both restriction of foreign DNA and recombinational DNA repair. Switching enzyme function from the destructive antiviral state to the productive recombinational state is regulated by the recombination hotspot, χ (5'-GCTGGTGG-3'). Recognition of χ is unique in that it is recognized as a specific sequence within single-stranded DNA (ssDNA) during DNA translocation and unwinding by RecBCD. The molecular determinants of χ recognition and the subsequent alteration in function are unknown. Consequently, we mutated residues within the RecC subunit that comprise a channel where ssDNA is thought to be scanned for a χ sequence. These mutants were characterized in vivo with regard to χ recognition, UV-sensitivity, phage degradation, and recombination proficiency. Of 38 residues mutated, 11 were previously undescribed mutations that altered χ recognition. The mutants fell into two classes: five that failed to respond to χ, and six that suggested a relaxed specificity for χ recognition. The location of the first set of mutations defines a recognition structure responsible for sequence-specific binding of ssDNA. The second set defines a highly conserved structure, linked to the recognition structure, which we hypothesize regulates conversion of RecBCD from a molecular machine that destroys DNA to one that repairs it. These findings offer insight into the evolution of enzymes with alternate χ recognition specificities.}, }
@article {pmid22603793, year = {2012}, author = {Yang, L and Handa, N and Liu, B and Dillingham, MS and Wigley, DB and Kowalczykowski, SC}, title = {Alteration of χ recognition by RecBCD reveals a regulated molecular latch and suggests a channel-bypass mechanism for biological control.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, number = {23}, pages = {8907-8912}, pmid = {22603793}, issn = {1091-6490}, support = {12799/CRUK_/Cancer Research UK/United Kingdom ; R01 GM041347/GM/NIGMS NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; GM41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Repair/*genetics/physiology ; DNA, Single-Stranded/genetics/*metabolism ; Escherichia coli/*enzymology ; Escherichia coli Proteins/genetics ; Exodeoxyribonuclease V/genetics/*metabolism ; *Models, Molecular ; Mutagenesis, Site-Directed ; Protein Structure, Secondary/genetics/physiology ; Regulatory Sequences, Nucleic Acid/*genetics ; Substrate Specificity ; }, abstract = {The RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at double-stranded DNA breaks. In Escherichia coli, its activities are regulated by the octameric recombination hotspot, χ (5'-GCTGGTGG), which is read as a single-stranded DNA sequence while the enzyme is unwinding DNA at over ∼1,000 bp/s. Previous studies implicated the RecC subunit as the "χ-scanning element" in this process. Site-directed mutagenesis and phenotypic analyses identified residues in RecC responsible for χ recognition [Handa N, et al., (2012) Proc Natl Acad Sci USA, 10.1073/pnas.1206076109]. The genetic analyses revealed two classes of mutants. Here we use ensemble and single-molecule criteria to biochemically establish that one class of mutants (type 1) has lost the capacity to recognize χ (lost-recognition), whereas the second class (type 2) has a lowered specificity for recognition (relaxed-specificity). The relaxed-specificity mutants still recognize canonical χ, but they have gained the capacity to precociously recognize single-nucleotide variants of χ. Based on the RecBCD structure, these mutant classes define an α-helix responsible for χ recognition that is allosterically coupled to a structural latch. When opened, we propose that the latch permits access to an alternative exit channel for the single-stranded DNA downstream of χ, thereby avoiding degradation by the nuclease domain. These findings provide a unique perspective into the mechanism by which recognition of a single-stranded DNA sequence switches the translocating RecBCD from a destructive nuclease to a constructive component of recombinational DNA repair.}, }
@article {pmid22307084, year = {2012}, author = {Saikrishnan, K and Yeeles, JT and Gilhooly, NS and Krajewski, WW and Dillingham, MS and Wigley, DB}, title = {Insights into Chi recognition from the structure of an AddAB-type helicase-nuclease complex.}, journal = {The EMBO journal}, volume = {31}, number = {6}, pages = {1568-1578}, pmid = {22307084}, issn = {1460-2075}, support = {12799/CRUK_/Cancer Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; }, mesh = {Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/chemistry/genetics/metabolism ; Binding Sites ; DNA Breaks, Double-Stranded ; DNA Helicases/*chemistry/genetics/metabolism ; DNA Repair ; DNA, Bacterial/chemistry/genetics/metabolism ; DNA, Single-Stranded/genetics ; DNA-Binding Proteins/chemistry/genetics/metabolism ; Deoxyribonucleases/*chemistry/genetics/metabolism ; Exodeoxyribonuclease V/chemistry/genetics/metabolism ; Exodeoxyribonucleases/*chemistry/genetics/metabolism ; Homologous Recombination ; Models, Molecular ; Mutation ; Protein Binding ; Protein Structure, Tertiary ; }, abstract = {In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi-recognition site in an inactivated helicase domain of the AddB subunit. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence-specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe-S cluster in protein stability and DNA binding.}, }
@article {pmid20852646, year = {2010}, author = {Wu, CG and Bradford, C and Lohman, TM}, title = {Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor.}, journal = {Nature structural &amp; molecular biology}, volume = {17}, number = {10}, pages = {1210-1217}, pmid = {20852646}, issn = {1545-9985}, support = {R01 GM045948-19/GM/NIGMS NIH HHS/United States ; R01 GM045948-18S1/GM/NIGMS NIH HHS/United States ; R01 GM045948-20/GM/NIGMS NIH HHS/United States ; GM045948/GM/NIGMS NIH HHS/United States ; R01 GM045948/GM/NIGMS NIH HHS/United States ; R01 GM045948-18/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphatases/chemistry/*physiology ; Adenosine Triphosphate/*metabolism ; DNA Breaks, Double-Stranded ; DNA Helicases/chemistry/*physiology ; DNA Repair/*physiology ; DNA, Bacterial/metabolism ; Escherichia coli/enzymology ; Escherichia coli Proteins/chemistry/*physiology ; Exodeoxyribonuclease V/chemistry/*physiology ; Models, Molecular ; Molecular Motor Proteins/physiology ; Multienzyme Complexes ; Protein Binding ; Protein Conformation ; Protein Subunits ; Recombinant Fusion Proteins/chemistry/physiology ; Structure-Activity Relationship ; }, abstract = {E. coli RecBCD is a DNA helicase with two ATPase motors (RecB, a 3'→5' translocase, and RecD, a 5'→3' translocase) that function in repair of double-stranded DNA breaks. The RecBC heterodimer, with only the RecB motor, remains a processive helicase. Here we examined RecBC translocation along single-stranded DNA (ssDNA). Notably, we found RecBC to have two translocase activities: the primary translocase moves 3'→5', whereas the secondary translocase moves RecBC along the opposite strand of a forked DNA at a similar rate. The secondary translocase is insensitive to the ssDNA backbone polarity, and we propose that it may fuel RecBCD translocation along double-stranded DNA ahead of the unwinding fork and ensure that the unwound single strands move through RecBCD at the same rate after interaction with a crossover hot-spot indicator (Chi) sequence.}, }
@article {pmid19254546, year = {2009}, author = {Fan, HF and Li, HW}, title = {Studying RecBCD helicase translocation along Chi-DNA using tethered particle motion with a stretching force.}, journal = {Biophysical journal}, volume = {96}, number = {5}, pages = {1875-1883}, pmid = {19254546}, issn = {1542-0086}, mesh = {Adenosine Triphosphate/metabolism ; Biophysics/methods ; DNA Helicases/*chemistry ; DNA, Bacterial/*chemistry/metabolism ; Escherichia coli/enzymology ; Exodeoxyribonuclease V/*chemistry/*metabolism ; Linear Models ; Streptavidin ; }, abstract = {Escherichia coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments showed that RecBCD recognizes an 8 nt DNA sequence, chi, and lowers its unwinding rate afterward under saturating ATP condition. We have developed a single-molecule force-tethered particle motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in detail. In the FTPM experiment, a stretching force is applied to the DNA-bead complex that suppresses the bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean-square displacement of the bead's Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties of conventional TPM experiments, such as nonlinearity and low resolution of long DNA substrates. The FTPM method offers the best resolution in the presence of only a small stretching force (0.20 pN). We used the FTPM method to investigate RecBCD helicase motion along 4.1 kb long chi-containing duplex DNA molecules, and observed that the translocation rate of RecBCD changes after the chi sequence under limited ATP concentrations. This suggests that chi recognition by RecBCD does not require saturating ATP conditions, contrary to what was previously reported.}, }
@article {pmid19052323, year = {2008}, author = {Dillingham, MS and Kowalczykowski, SC}, title = {RecBCD enzyme and the repair of double-stranded DNA breaks.}, journal = {Microbiology and molecular biology reviews : MMBR}, volume = {72}, number = {4}, pages = {642-71, Table of Contents}, pmid = {19052323}, issn = {1098-5557}, mesh = {*DNA Breaks, Double-Stranded ; *DNA Repair ; DNA, Bacterial/chemistry/genetics ; Escherichia coli/*enzymology/genetics ; Exodeoxyribonuclease V/genetics/*metabolism ; Exodeoxyribonucleases/metabolism ; Recombination, Genetic ; }, abstract = {The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5' strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.}, }
@article {pmid18847457, year = {2008}, author = {Portakal, O and Doğan, P}, title = {Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli.}, journal = {BMC biochemistry}, volume = {9}, number = {}, pages = {27}, pmid = {18847457}, issn = {1471-2091}, mesh = {Alleles ; Base Sequence ; DNA, Bacterial/metabolism ; Escherichia coli/*enzymology/genetics/metabolism ; Escherichia coli Proteins/*genetics/*metabolism ; Exodeoxyribonuclease V/*genetics/metabolism/physiology ; Haploidy ; Models, Biological ; Molecular Sequence Data ; Mutation ; Phenotype ; Recombinant Fusion Proteins/genetics/metabolism ; Recombination, Genetic ; }, abstract = {BACKGROUND: recD, located between recB and argA, encodes the smallest polypeptide (60 kDa) of the heterotrimeric enzyme RecBCD in Escherichia coli. RecD is a 5'-3' helicase and is required for the nuclease activity of RecBCD and for tight binding to dsDNA ends. Here, we have tested the hypothesis that RecD regulates the structure and activities of RecBCD, including RecA loading.<br><br>RESULTS: To characterize its regulatory functions, recD was genetically fused to recB through deletion and substitution mutations. The recB-recD fusion led to a decreased amount of the heterotrimer. Both fusion mutants proved to be recombination proficient, viable and resistant to DNA damaging agents, and to have DNA unwinding, ATP-dependent dsDNA exonuclease and Chi genetic activities.<br><br>CONCLUSION: Our findings suggest that the recB-recD fusion may form a RecBD fusion protein and therefore affect RecD assembly, but this does not change the three-dimensional structure of the heterotrimer.}, }
@article {pmid18079176, year = {2007}, author = {Amundsen, SK and Taylor, AF and Reddy, M and Smith, GR}, title = {Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots.}, journal = {Genes &amp; development}, volume = {21}, number = {24}, pages = {3296-3307}, pmid = {18079176}, issn = {0890-9369}, support = {R01 GM031693/GM/NIGMS NIH HHS/United States ; R56 GM031693/GM/NIGMS NIH HHS/United States ; GM031693/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA/metabolism ; DNA Helicases/genetics/*metabolism ; Escherichia coli/enzymology ; Mutation ; *Signal Transduction ; }, abstract = {The Escherichia coli RecBCD helicase-nuclease, a paradigm of complex protein machines, initiates homologous genetic recombination and the repair of broken DNA. Starting at a duplex end, RecBCD unwinds DNA with its fast RecD helicase and slower RecB helicase on complementary strands. Upon encountering a Chi hot spot (5'-GCTGGTGG-3'), the enzyme produces a new 3' single-strand end and loads RecA protein onto it, but how Chi regulates RecBCD is unknown. We report a new class of mutant RecBCD enzymes that cut DNA at novel positions that depend on the DNA substrate length and that are strictly correlated with the RecB:RecD helicase rates. We conclude that in the mutant enzymes when RecD reaches the DNA end, it signals RecB's nuclease domain to cut the DNA. As predicted by this interpretation, the mutant enzymes cut closer to the entry point on DNA when unwinding is blocked by another RecBCD molecule traveling in the opposite direction. Furthermore, when RecD is slowed by a mutation altering its ATPase site such that RecB reaches the DNA end before RecD does, the length-dependent cuts are abolished. These observations lead us to hypothesize that, in wild-type RecBCD enzyme, Chi is recognized by RecC, which then signals RecD to stop, which in turn signals RecB to cut the DNA and load RecA. We discuss support for this "signal cascade" hypothesis and tests of it. Intersubunit signaling may regulate other complex protein machines.}, }
@article {pmid18022364, year = {2007}, author = {Spies, M and Amitani, I and Baskin, RJ and Kowalczykowski, SC}, title = {RecBCD enzyme switches lead motor subunits in response to chi recognition.}, journal = {Cell}, volume = {131}, number = {4}, pages = {694-705}, pmid = {18022364}, issn = {0092-8674}, support = {R01 GM041347-13/GM/NIGMS NIH HHS/United States ; R01 GM041347/GM/NIGMS NIH HHS/United States ; R01 GM041347-12/GM/NIGMS NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; R01 GM041347-14/GM/NIGMS NIH HHS/United States ; }, mesh = {*DNA Repair ; DNA, Bacterial/chemistry/genetics/metabolism ; Escherichia coli Proteins/chemistry/genetics/metabolism ; Exodeoxyribonuclease V/*chemistry/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Molecular Motor Proteins/chemistry/genetics/metabolism ; Mutation ; Nucleic Acid Conformation ; Protein Subunits/*chemistry/genetics/*metabolism ; *Recombination, Genetic ; Single-Strand Specific DNA and RNA Endonucleases/metabolism ; }, abstract = {RecBCD is a DNA helicase comprising two motor subunits, RecB and RecD. Recognition of the recombination hotspot, chi, causes RecBCD to pause and reduce translocation speed. To understand this control of translocation, we used single-molecule visualization to compare RecBCD to the RecBCD(K177Q) mutant with a defective RecD motor. RecBCD(K177Q) paused at chi but did not change its translocation velocity. RecBCD(K177Q) translocated at the same rate as the wild-type post-chi enzyme, implicating RecB as the lead motor after chi. P1 nuclease treatment eliminated the wild-type enzyme's velocity changes, revealing a chi-containing ssDNA loop preceding chi recognition and showing that RecD is the faster motor before chi. We conclude that before chi, RecD is the lead motor but after chi, the slower RecB motor leads, implying a switch in motors at chi. We suggest that degradation of foreign DNA needs fast translocation, whereas DNA repair uses slower translocation to coordinate RecA loading onto ssDNA.}, }
@article {pmid18022359, year = {2007}, author = {Wigley, DB}, title = {RecBCD: the supercar of DNA repair.}, journal = {Cell}, volume = {131}, number = {4}, pages = {651-653}, doi = {10.1016/j.cell.2007.11.004}, pmid = {18022359}, issn = {0092-8674}, mesh = {*DNA Repair ; Exodeoxyribonuclease V/chemistry/*metabolism ; Models, Molecular ; Protein Structure, Quaternary ; Protein Subunits/chemistry/metabolism ; Recombination, Genetic ; }, abstract = {The DNA helicase RecBCD pauses when it reaches recombination hotspots known as Chi sites and then proceeds at a slower speed of translocation than before Chi recognition. Reporting in this issue, Spies et al. (2007) now show that this reduction in translocation velocity occurs when RecBCD changes which of its two motor subunits is in the lead.}, }
@article {pmid17544443, year = {2007}, author = {Court, R and Cook, N and Saikrishnan, K and Wigley, D}, title = {The crystal structure of lambda-Gam protein suggests a model for RecBCD inhibition.}, journal = {Journal of molecular biology}, volume = {371}, number = {1}, pages = {25-33}, doi = {10.1016/j.jmb.2007.05.037}, pmid = {17544443}, issn = {0022-2836}, support = {P41 RR-01081/RR/NCRR NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*metabolism ; Crystallography, X-Ray ; DNA/metabolism ; DNA-Binding Proteins ; Dimerization ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/chemistry/genetics/metabolism ; Exodeoxyribonuclease V/*antagonists &amp; inhibitors/chemistry/genetics/metabolism ; Models, Molecular ; Molecular Sequence Data ; Protein Binding ; *Protein Conformation ; Viral Proteins/*chemistry/genetics/*metabolism ; }, abstract = {In Escherichia coli, RecBCD processes double-stranded DNA breaks during the initial stages of homologous recombination. RecBCD contains helicase and nuclease activities, and unwinds and digests the blunt-ended DNA until a specific eight-nucleotide sequence, Chi, is encountered. Chi modulates the nuclease activity of RecBCD and results in a resected DNA end, which is a substrate for RecA during subsequent steps in recombination. RecBCD also acts as a defence mechanism against bacteriophage infection by digesting linear viral DNA present during virus replication or resulting from the action of restriction endonucleases. To avoid this fate, bacteriophage lambda encodes the gene Gam whose product is an inhibitor of RecBCD. Gam has been shown to bind to RecBCD and inhibit its helicase and nuclease activities. We show that Gam inhibits RecBCD by preventing it from binding DNA. We have solved the crystal structure of Gam from two different crystal forms. Using the published crystal structure of RecBCD in complex with DNA we suggest models for the molecular mechanism of Gam-mediated inhibition of RecBCD. We also propose that Gam could be a mimetic of single-stranded, and perhaps also double-stranded, DNA.}, }
@article {pmid17270364, year = {2007}, author = {Arakawa, K and Uno, R and Nakayama, Y and Tomita, M}, title = {Validating the significance of genomic properties of Chi sites from the distribution of all octamers in Escherichia coli.}, journal = {Gene}, volume = {392}, number = {1-2}, pages = {239-246}, doi = {10.1016/j.gene.2006.12.022}, pmid = {17270364}, issn = {0378-1119}, mesh = {*Chromosome Mapping ; DNA Replication ; Escherichia coli/*genetics ; *Genetic Markers ; Models, Theoretical ; Open Reading Frames ; RNA, Messenger/genetics ; Recombination, Genetic/genetics ; Statistical Distributions ; }, abstract = {Chi sites (5'-GCTGGTGG-3') are homologous recombinational hotspot octamer sequences, which attenuate the exonuclease activity of RecBCD in Escherichia coli. They are overrepresented in the genome (1008 occurrences), preferentially located within coding regions (98%), oriented in the direction of replication (75%), and occur most commonly on the mRNA-synonymous sense strand of the double helix (79%). Previous statistical studies of the genome sequence suggested that these genomic properties of Chi sites appear to be related to their role in recombinational repair and therefore to replication and transcription. In this study, we employ three mathematical models to predict the properties of Chi sites from single nucleotide and multi-nucleotide compositions, and validate them statistically using the distribution of all octamer sequences in the entire genome, or exclusively within ORFs. The model based on the overall distribution of all octamers provided better predictions than the single nucleotide composition model, and the ORF and sense strand preference of Chi sites were shown to be within the standard deviation of all octamers. In contrast, the orientation bias of the Chi sites in the direction of replication was significant, although the bias was not as pronounced as with the single nucleotide composition model, suggesting a selective pressure related to the role of RecBCD in replication.}, }
@article {pmid17141804, year = {2007}, author = {Handa, N and Kowalczykowski, SC}, title = {A RecA mutant, RecA(730), suppresses the recombination deficiency of the RecBC(1004)D-chi* interaction in vitro and in vivo.}, journal = {Journal of molecular biology}, volume = {365}, number = {5}, pages = {1314-1325}, pmid = {17141804}, issn = {0022-2836}, support = {R01 GM041347/GM/NIGMS NIH HHS/United States ; R01 GM062653/GM/NIGMS NIH HHS/United States ; R37 GM062653/GM/NIGMS NIH HHS/United States ; GM 41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA, Bacterial/metabolism/radiation effects ; DNA, Single-Stranded/metabolism/radiation effects ; DNA-Binding Proteins/metabolism ; Escherichia coli/*enzymology/*genetics/radiation effects ; Escherichia coli Proteins/metabolism ; Exodeoxyribonuclease V/*metabolism ; Mutant Proteins/*metabolism ; Mutation/genetics ; Nucleoproteins/metabolism ; Protein Binding/radiation effects ; Protein Structure, Quaternary/radiation effects ; Rec A Recombinases/*metabolism ; *Recombination, Genetic/radiation effects ; Regulatory Sequences, Nucleic Acid/*genetics/radiation effects ; Ultraviolet Rays ; }, abstract = {In Escherichia coli, homologous recombination initiated at double-stranded DNA breaks requires the RecBCD enzyme, a multifunctional heterotrimeric complex that possesses processive helicase and exonuclease activities. Upon encountering the DNA regulatory sequence, chi, the enzymatic properties of RecBCD enzyme are altered. Its helicase activity is reduced, the 3'-->5'nuclease activity is attenuated, the 5'-->3' nuclease activity is up-regulated, and it manifests an ability to load RecA protein onto single-stranded DNA. The net result of these changes is the production of a highly recombinogenic structure known as the presynaptic filament. Previously, we found that the recC1004 mutation alters chi-recognition so that this mutant enzyme recognizes an altered chi sequence, chi*, which comprises seven of the original nucleotides in chi, plus four novel nucleotides. Although some consequences of this mutant enzyme-mutant chi interaction could be detected in vivo and in vitro, stimulation of recombination in vivo could not. To resolve this seemingly contradictory observation, we examined the behavior of a RecA mutant, RecA(730), that displays enhanced biochemical activity in vitro and possesses suppressor function in vivo. We show that the recombination deficiency of the RecBC(1004)D-chi* interaction can be overcome by the enhanced ability of RecA(730) to assemble on single-stranded DNA in vitro and in vivo. These data are consistent with findings showing that the loading of RecA protein by RecBCD is necessary in vivo, and they show that RecA proteins with enhanced single-stranded DNA-binding capacity can partially bypass the need for RecBCD-mediated loading.}, }
@article {pmid17110484, year = {2007}, author = {Amundsen, SK and Smith, GR}, title = {Chi hotspot activity in Escherichia coli without RecBCD exonuclease activity: implications for the mechanism of recombination.}, journal = {Genetics}, volume = {175}, number = {1}, pages = {41-54}, pmid = {17110484}, issn = {0016-6731}, support = {R01 GM031693/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/enzymology/genetics/growth &amp; development ; Crosses, Genetic ; DNA, Bacterial/genetics/metabolism ; Escherichia coli/*enzymology/genetics ; Exodeoxyribonuclease V/genetics/*metabolism ; Mutation ; Plasmids ; Recombination, Genetic/*genetics ; }, abstract = {The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (5'-GCTGGTGG-3'). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and lambda phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.}, }
@article {pmid16901504, year = {2006}, author = {Wong, CJ and Rice, RL and Baker, NA and Ju, T and Lohman, TM}, title = {Probing 3'-ssDNA loop formation in E. coli RecBCD/RecBC-DNA complexes using non-natural DNA: a model for "Chi" recognition complexes.}, journal = {Journal of molecular biology}, volume = {362}, number = {1}, pages = {26-43}, doi = {10.1016/j.jmb.2006.07.016}, pmid = {16901504}, issn = {0022-2836}, support = {R01 GM045948/GM/NIGMS NIH HHS/United States ; GM45948/GM/NIGMS NIH HHS/United States ; }, mesh = {*DNA, Single-Stranded/chemistry/metabolism ; Escherichia coli Proteins/*chemistry/metabolism ; Exodeoxyribonuclease V/*chemistry/metabolism ; Macromolecular Substances ; Models, Molecular ; Molecular Structure ; *Nucleic Acid Conformation ; Protein Conformation ; Thermodynamics ; }, abstract = {The equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends containing varying lengths of polyethylene glycol (PEG) spacers within pre-formed 3'-single-stranded (ss) DNA ((dT)n) tails was studied. These studies were designed to test a previous proposal that the 3'-(dT)n tail can be looped out upon binding RecBC and RecBCD for 3'-ssDNA tails with n>or=6 nucleotides. Equilibrium binding of protein to unlabeled DNA substrates with ends containing PEG-substituted 3'-ssDNA tails was examined by competition with a Cy3-labeled reference DNA which undergoes a Cy3 fluorescence enhancement upon protein binding. We find that the binding affinities of both RecBC and RecBCD for a DNA end are unaffected upon substituting PEG for the ssDNA between the sixth and the final two nucleotides of the 3'-(dT)n tail. However, placing PEG at the end of the 3'-(dT)n tail increases the binding affinities to their maximum values (i.e. the same as binding constants for RecBC or RecBCD to a DNA end with only a 3'-(dT)6 tail). Equilibrium binding studies of a RecBC mutant containing a nuclease domain deletion, RecB(Deltanuc)C, suggest that looping of the 3'-tail (when n>or=6 nucleotides) occurs even in the absence of the RecB nuclease domain, although the nuclease domain stabilizes such loop formation. Computer modeling of the RecBCD-DNA complexes suggests that the loop in the 3'-ssDNA tail may form at the RecB/RecC interface. Based on these results we suggest a model for how a loop in the 3'-ssDNA tail might form upon encounter of a "Chi" recognition sequence during unwinding of DNA by the RecBCD helicase.}, }
@article {pmid16887143, year = {2006}, author = {Dziegielewska, B and Beerman, TA and Bianco, PR}, title = {Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.}, journal = {Journal of molecular biology}, volume = {361}, number = {5}, pages = {898-919}, doi = {10.1016/j.jmb.2006.06.068}, pmid = {16887143}, issn = {0022-2836}, mesh = {Adenosine Triphosphate/metabolism ; Allosteric Site/drug effects ; Anthraquinones/chemistry/pharmacology ; Antineoplastic Agents, Alkylating/chemistry/*pharmacology ; Benzofurans ; Catalysis/drug effects ; Cyclohexanecarboxylic Acids/chemistry/pharmacology ; Cyclohexenes ; DNA/*chemistry ; DNA Helicases/antagonists &amp; inhibitors ; Duocarmycins ; Escherichia coli/*enzymology ; Exodeoxyribonuclease V/*antagonists &amp; inhibitors/metabolism ; Fluorescence ; Hydrolysis/drug effects ; Indoles/chemistry/pharmacology ; Models, Molecular ; Nucleic Acid Conformation/drug effects ; Recombination, Genetic/drug effects ; }, abstract = {To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.}, }
@article {pmid16854534, year = {2006}, author = {Uno, R and Nakayama, Y and Tomita, M}, title = {Over-representation of Chi sequences caused by di-codon increase in Escherichia coli K-12.}, journal = {Gene}, volume = {380}, number = {1}, pages = {30-37}, doi = {10.1016/j.gene.2006.05.013}, pmid = {16854534}, issn = {0378-1119}, mesh = {Base Composition ; Base Sequence ; Chi-Square Distribution ; Codon/genetics ; DNA, Bacterial/chemistry/*genetics ; Escherichia coli/genetics ; Escherichia coli K12/*genetics/metabolism ; Escherichia coli O157/genetics ; Exodeoxyribonuclease V/metabolism ; Genes, Bacterial ; Reading Frames ; Recombination, Genetic ; Species Specificity ; }, abstract = {Chi sequences (5'-GCTGGTGG-3') are cis-acting 8 bp sequence elements that enhance homologous recombination promoted by the RecBCD pathway in Escherichia coli. The genome of E. coli K-12 MG1655 contains 1009 Chi sequences and this frequency far exceeds the expected value for occurrence of an 8 bp sequence in a genome of this size. It is generally thought that the over-representation of Chi sequences indicates that they have been selected for during evolution because of their function in recombination. The genes from three E. coli strains (K-12, O157 and CFT) were classified into three categories (island, match to other E. coli, and backbone). Island genes have a different base composition and codon usage in comparison with those in the backbone genes, therefore they were relatively new and not yet adapted to the base composition patterns and codon usage typical of the recipient genome. The over-representation of Chi sequences was examined by comparing Chi frequencies and codon frequencies between island and backbone genes. The difference in the CTGGTG di-codon frequency between the backbone and island genes was correlated with the frequency of Chi sequences which were translated in the Leu-Val (-G/CTG/GTG/G-) reading frame in the K-12 strain. These results suggest that the main reading frame of Chi sequences increased as a result of the di-codon CTG-GTG increasing under a genome-wide pressure for adapting to the codon usage and base composition of the E. coli K-12 strain, and that the RecBCD recombinase might adjust its recognition sequence to a frequently occurring oligomer such as G-CTG-GTG-G.}, }
@article {pmid16632468, year = {2006}, author = {Chédin, F and Handa, N and Dillingham, MS and Kowalczykowski, SC}, title = {The AddAB helicase/nuclease forms a stable complex with its cognate chi sequence during translocation.}, journal = {The Journal of biological chemistry}, volume = {281}, number = {27}, pages = {18610-18617}, doi = {10.1074/jbc.M600882200}, pmid = {16632468}, issn = {0021-9258}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; //Wellcome Trust/United Kingdom ; }, mesh = {Bacillus subtilis/*enzymology/genetics ; Bacterial Proteins/chemistry/genetics ; Binding Sites/genetics ; DNA Damage ; DNA Helicases/*chemistry/genetics ; DNA Repair ; Enzyme Activation/genetics ; Exodeoxyribonucleases/*chemistry/genetics ; Sequence Analysis, DNA ; Substrate Specificity ; Translocation, Genetic ; }, abstract = {The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (chi). Recognition of chi triggers attenuation of the 3'- to 5'-nuclease, which permits the generation of recombinogenic 3'-overhanging, single-stranded DNA (ssDNA), terminating at chi. Although the RecBCD enzyme briefly pauses at chi, no specific binding of RecBCD to chi during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate chi sequence (chi(Bs): 5'-AGCGG-3') during translocation. The binding of AddAB enzyme to the 3'-end of the chi(Bs)-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, chi(Bs)-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to chi(Bs) with high affinity only during translocation. Finally, protection of chi(Bs)-specific ssDNA is still observed when translocation occurs in the presence of competitor chi(Bs)-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to chi(Bs) is an integral part of the AddAB-chi(Bs) interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.}, }
@article {pmid16377056, year = {2006}, author = {Dermić, D and Dermić, E and Zahradka, D and Petranović, M and Lers, N}, title = {Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome.}, journal = {Biochimie}, volume = {88}, number = {3-4}, pages = {379-386}, doi = {10.1016/j.biochi.2005.11.003}, pmid = {16377056}, issn = {0300-9084}, mesh = {Bacteriophage T4/metabolism/pathogenicity ; Bacteriophage lambda/genetics/metabolism ; Chromosomes, Bacterial/genetics/metabolism/*radiation effects ; DNA Damage ; *DNA Repair ; DNA, Bacterial/genetics/*metabolism/radiation effects ; Escherichia coli/*genetics/metabolism/radiation effects ; Escherichia coli Proteins/genetics/*metabolism ; Exodeoxyribonuclease V/genetics/*metabolism ; *Gamma Rays ; Gene Expression Regulation, Bacterial/radiation effects ; Peptides/metabolism ; Recombination, Genetic ; Ultraviolet Rays ; }, abstract = {The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.}, }
@article {pmid16126227, year = {2005}, author = {Wong, CJ and Lucius, AL and Lohman, TM}, title = {Energetics of DNA end binding by E.coli RecBC and RecBCD helicases indicate loop formation in the 3'-single-stranded DNA tail.}, journal = {Journal of molecular biology}, volume = {352}, number = {4}, pages = {765-782}, doi = {10.1016/j.jmb.2005.07.056}, pmid = {16126227}, issn = {0022-2836}, support = {R01 GM045948/GM/NIGMS NIH HHS/United States ; 5 T32 GM08492/GM/NIGMS NIH HHS/United States ; GM45948/GM/NIGMS NIH HHS/United States ; }, mesh = {Base Sequence ; Carbocyanines/chemistry ; *DNA, Single-Stranded/chemistry/metabolism ; Escherichia coli Proteins/*metabolism ; Exodeoxyribonuclease V/*metabolism ; Fluorescent Dyes/chemistry ; Macromolecular Substances ; Molecular Sequence Data ; Molecular Structure ; *Nucleic Acid Conformation ; Protein Binding ; }, abstract = {We examined the equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends possessing pre-existing single-stranded (ss) DNA ((dT)(n)) tails varying in length (n=0 to 20 nucleotides) in order to determine the contributions of both the 3' and 5' single strands to the energetics of complex formation. Protein binding was monitored by the fluorescence enhancement of a reference DNA labeled at its end with a Cy3 fluorophore. Binding to unlabeled DNA was examined by competition titrations with the Cy3-labeled reference DNA. The affinities of both RecBC and RecBCD increase as the 3'-(dT)(n) tail length increases from zero to six nucleotides, but then decrease dramatically as the 3'-(dT)(n) tail length increases from six to 20 nucleotides. Isothermal titration calorimetry experiments with RecBC show that the binding enthalpy is negative and increases in magnitude with increasing 3'-(dT)(n) tail length up to n=6 nucleotides, but remains constant for n > or =6. Hence, the decrease in binding affinity for 3'-(dT)(n) tail lengths with n > or =6 is due to an unfavorable entropic contribution. RecBC binds optimally to duplex DNA with (dT)6 tails on both the 3' and 5'-ends while RecBCD prefers duplex DNA with 3'-(dT)6 and 5'-(dT)10 tails. These data suggest that both RecBC and RecBCD helicases can destabilize or "melt out" six base-pairs upon binding to a blunt DNA duplex end in the absence of ATP. These results also provide the first evidence that a loop in the 3'-ssDNA tail can form upon binding of RecBC or RecBCD with DNA duplexes containing a pre-formed 3'-ssDNA tail with n > or =6 nucleotides. Such loops may be representative of those hypothesized to form upon interaction of a Chi site contained within the unwound 3' ss-DNA tail with the RecC subunit during DNA unwinding.}, }
@article {pmid16041060, year = {2005}, author = {Spies, M and Dillingham, MS and Kowalczykowski, SC}, title = {Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme.}, journal = {The Journal of biological chemistry}, volume = {280}, number = {44}, pages = {37078-37087}, doi = {10.1074/jbc.M505521200}, pmid = {16041060}, issn = {0021-9258}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphatases/genetics/metabolism ; Adenosine Triphosphate/metabolism ; Binding Sites ; DNA Helicases/chemistry ; DNA, Bacterial/metabolism ; DNA, Single-Stranded/*metabolism ; Escherichia coli Proteins/*metabolism ; Exodeoxyribonuclease V/chemistry/*metabolism ; Gene Expression Regulation, Bacterial ; *Gene Expression Regulation, Enzymologic ; Mutagenesis, Site-Directed ; Mutation ; Protein Transport ; Rec A Recombinases/chemistry/*metabolism ; *Recombination, Genetic ; }, abstract = {RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. The enzyme is driven by two motor subunits, RecB and RecD, translocating on opposite single-strands of the DNA duplex. Here we provide evidence that, although both motor subunits can support the translocation activity for the enzyme, the activity of the RecB subunit is necessary for proper function of the enzyme both in vivo and in vitro. We demonstrate that the RecBCD(K177Q) enzyme, in which RecD helicase is disabled by mutation of the ATPase active site, complements recBCD deletion in vivo and displays all of the enzymatic activities that are characteristic of the wild-type enzyme in vitro. These include helicase and nuclease activities and the abilities to recognize the recombination hotspot chi and to coordinate the loading of RecA protein onto the ssDNA it produces. In contrast, the RecB(K29Q)CD enzyme, carrying a mutation in the ATPase site of RecB helicase, fails to complement recBCD deletion in vivo. We further show that even though RecB(K29Q)CD enzyme displays helicase and nuclease activities, its inability to translocate along the 3'-terminated strand results in the failure to recognize chi and to load RecA protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to chi, whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.}, }
@article {pmid15749023, year = {2005}, author = {Handa, N and Bianco, PR and Baskin, RJ and Kowalczykowski, SC}, title = {Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition.}, journal = {Molecular cell}, volume = {17}, number = {5}, pages = {745-750}, doi = {10.1016/j.molcel.2005.02.011}, pmid = {15749023}, issn = {1097-2765}, support = {R01 GM064745/GM/NIGMS NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; GM-64745/GM/NIGMS NIH HHS/United States ; }, mesh = {Biotinylation ; DNA/chemistry/metabolism ; Escherichia coli/*metabolism ; Escherichia coli Proteins/chemistry/*metabolism ; Exodeoxyribonuclease V/*metabolism ; Macromolecular Substances/chemistry ; Microscopy, Fluorescence/*methods ; Models, Genetic ; Nanostructures ; Plasmids/metabolism ; Protein Transport ; Recombination, Genetic ; Time Factors ; }, abstract = {In Escherichia coli, chi (5'-GCTGGTGG-3') is a recombination hotspot recognized by the RecBCD enzyme. Recognition of chi reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by chi interaction has been "ejection" of the RecD motor from the holoenzyme at chi. To test this proposal, we visualized individual RecBCD molecules labeled via RecD with a fluorescent nanoparticle. We could directly see these labeled, single molecules of RecBCD moving at up to 1835 bp/s (approximately 0.6 microm/s). Those enzymes translocated to chi, paused, and continued at reduced velocity, without loss of RecD. We conclude that chi interaction induces a conformational change, resulting from binding of chi to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.}, }
@article {pmid15538360, year = {2004}, author = {Singleton, MR and Dillingham, MS and Gaudier, M and Kowalczykowski, SC and Wigley, DB}, title = {Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks.}, journal = {Nature}, volume = {432}, number = {7014}, pages = {187-193}, doi = {10.1038/nature02988}, pmid = {15538360}, issn = {1476-4687}, mesh = {Crystallization ; Crystallography, X-Ray ; DNA/chemistry/genetics/*metabolism ; *DNA Damage ; DNA Helicases/chemistry/metabolism ; *DNA Repair ; Endonucleases/chemistry/metabolism ; Escherichia coli/*enzymology ; Exodeoxyribonuclease V/*chemistry/*metabolism ; Models, Molecular ; Multienzyme Complexes/chemistry/metabolism ; Nucleic Acid Conformation ; Protein Conformation ; Structure-Activity Relationship ; Substrate Specificity ; }, abstract = {RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.}, }
@article {pmid15254271, year = {2004}, author = {Kulkarni, A and Julin, DA}, title = {Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides.}, journal = {Nucleic acids research}, volume = {32}, number = {12}, pages = {3672-3682}, pmid = {15254271}, issn = {1362-4962}, support = {R01 GM039777/GM/NIGMS NIH HHS/United States ; GM39777/GM/NIGMS NIH HHS/United States ; }, mesh = {Base Sequence ; DNA/metabolism ; DNA Helicases/metabolism ; DNA, Single-Stranded/chemistry/*pharmacology ; Enzyme Inhibitors/*chemistry/pharmacology ; Escherichia coli/*enzymology ; Escherichia coli Proteins/metabolism ; Exodeoxyribonuclease V/*metabolism ; Oligodeoxyribonucleotides/chemistry/*pharmacology ; Regulatory Sequences, Nucleic Acid ; }, abstract = {RecBCD is an ATP-dependent helicase and exonuclease which generates 3' single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5'-GCTGGTGG-3') in double-stranded DNA (ds DNA), an event critical to the generation of the 3'-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Ch+) or a single base change that abolishes the Chi sequence (Chi(o)), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi+ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi(o) oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.}, }
@article {pmid13678587, year = {2003}, author = {Spies, M and Bianco, PR and Dillingham, MS and Handa, N and Baskin, RJ and Kowalczykowski, SC}, title = {A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase.}, journal = {Cell}, volume = {114}, number = {5}, pages = {647-654}, doi = {10.1016/s0092-8674(03)00681-0}, pmid = {13678587}, issn = {0092-8674}, support = {R01 GM064745/GM/NIGMS NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; GM-64745/GM/NIGMS NIH HHS/United States ; }, mesh = {Biological Transport ; DNA/*metabolism/*physiology ; DNA, Single-Stranded/metabolism ; Dimerization ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*chemistry/*physiology ; Kinetics ; Microscopy, Fluorescence ; Models, Biological ; Models, Genetic ; Protein Transport ; *Recombination, Genetic ; Time Factors ; }, abstract = {RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. Several of its activities are regulated by the DNA sequence chi (5'-GCTGGTGG-3'), which is recognized in cis by the translocating enzyme. When RecBCD enzyme encounters chi, the intensity and polarity of its nuclease activity are changed, and the enzyme gains the ability to load RecA protein onto the chi-containing, unwound single-stranded DNA. Here, we show that interaction with chi also affects translocation by RecBCD enzyme. By observing translocation of individual enzymes along single molecules of DNA, we could see RecBCD enzyme pause precisely at chi. Furthermore, and more unexpectedly, after pausing at chi, the enzyme continues translocating but at approximately one-half the initial rate. We propose that interaction with chi results in an enzyme in which one of the two motor subunits, likely the RecD motor, is uncoupled from the holoenzyme to produce the slower translocase.}, }
@article {pmid12203773, year = {2002}, author = {Kolomietz, E and Meyn, MS and Pandita, A and Squire, JA}, title = {The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors.}, journal = {Genes, chromosomes &amp; cancer}, volume = {35}, number = {2}, pages = {97-112}, doi = {10.1002/gcc.10111}, pmid = {12203773}, issn = {1045-2257}, mesh = {Alu Elements/*physiology ; Animals ; *Chromosome Aberrations ; Humans ; Multigene Family/*physiology ; Neoplasms/*genetics/pathology ; Recurrence ; }, abstract = {There is increasing evidence for the involvement of repetitive DNA sequences as facilitators of some of the recurrent chromosomal rearrangements observed in human tumors. The high densities of repetitive DNA, such as Alu elements, at some chromosomal translocation breakpoint regions has led to the suggestion that these sequences could provide hot spots for homologous recombination, and could mediate the translocation process and elevate the likelihood of other types of chromosomal rearrangements taking place. The Alu core sequence itself has been suggested to promote DNA strand exchange and genomic rearrangement, and it has striking sequence similarity to chi (which has been shown to stimulate recBCD-mediated recombination in Escherichia coli). Alu repeats have been shown to be involved in the generation of many constitutional gene mutations in meiotic cells, attributed to unequal homologous recombination and consequent deletions and/or duplication events. It has recently been demonstrated that similar deletion events can take place in neoplasia because several types of leukemia-associated chromosomal rearrangements frequently have submicroscopic deletions immediately adjacent to the translocation breakpoint regions. Significantly, these types of deletions appear to be more likely to take place when the regions subject to rearrangement contain a high density of Alu repeats. With the completion of the Human Genome Project, it will soon be possible to create more comprehensive maps of the distribution and densities of repetitive sequences, such as Alu, throughout the genome. Such maps will offer unique insights into the relative distribution of cancer translocation breakpoints and the localization of clusters of repetitive DNA.}, }
@article {pmid12072448, year = {2002}, author = {Amundsen, SK and Taylor, AF and Smith, GR}, title = {A domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme.}, journal = {Genetics}, volume = {161}, number = {2}, pages = {483-492}, pmid = {12072448}, issn = {0016-6731}, support = {GM-31693/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA, Bacterial/metabolism ; Escherichia coli/*enzymology/genetics ; Escherichia coli Proteins/metabolism ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/chemistry/genetics/*metabolism ; Holoenzymes/chemistry/metabolism ; Mutation ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Rec A Recombinases/metabolism ; Sequence Deletion ; }, abstract = {The heterotrimeric RecBCD enzyme of Escherichia coli is required for the major pathway of double-strand DNA break repair and genetic exchange. Assembled as a heterotrimer, the enzyme has potent nuclease and helicase activity. Analysis of recC nonsense and deletion mutations revealed that the C terminus of RecC is required for assembly of the RecD subunit into RecBCD holoenzyme but not for recombination proficiency; the phenotype of these mutations mimics that of recD deletion mutations. Partial proteolysis of purified RecC polypeptide yielded a C-terminal fragment that corresponds to the RecD-interaction domain. RecD is essential for nuclease activity, regulation by the recombination hotspot Chi, and high affinity for DNA ends. The RecC-RecD interface thus appears critical for the regulation of RecBCD enzyme via the assembly and, we propose, disassembly or conformational change of the RecD subunit.}, }
@article {pmid11532156, year = {2001}, author = {Jockovich, ME and Myers, RS}, title = {Nuclease activity is essential for RecBCD recombination in Escherichia coli.}, journal = {Molecular microbiology}, volume = {41}, number = {4}, pages = {949-962}, doi = {10.1046/j.1365-2958.2001.02573.x}, pmid = {11532156}, issn = {0950-382X}, mesh = {Bacterial Proteins/genetics/physiology ; Bacteriophage lambda/genetics/physiology ; DNA Repair/radiation effects ; DNA Replication/radiation effects ; DNA-Binding Proteins/genetics/physiology ; Escherichia coli/*enzymology/*genetics/radiation effects/virology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/chemistry/genetics/*metabolism ; Genotype ; Models, Genetic ; Mutagenesis/radiation effects ; *Recombination, Genetic/radiation effects ; Substrate Specificity ; Ultraviolet Rays ; }, abstract = {RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double-stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of chi recombination hot-spots. These paradoxical roles are accommodated by chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that chi converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5' --> 3' exonuclease activity is essential for recombination by RecBCD at chi recombination hot-spots and at dsDNA ends in recD mutants. In the absence of RecB-dependent nuclease function, recombination becomes entirely dependent on the 5' --> 3' single-stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).}, }
@article {pmid11395472, year = {2001}, author = {Quiberoni, A and Biswas, I and El Karoui, M and Rezaïki, L and Tailliez, P and Gruss, A}, title = {In vivo evidence for two active nuclease motifs in the double-strand break repair enzyme RexAB of Lactococcus lactis.}, journal = {Journal of bacteriology}, volume = {183}, number = {13}, pages = {4071-4078}, pmid = {11395472}, issn = {0021-9193}, mesh = {Amino Acid Motifs ; Amino Acid Sequence ; Catalytic Domain/genetics ; Conserved Sequence ; DNA Helicases/*metabolism ; *DNA Repair ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Lactococcus lactis/*enzymology/*genetics ; Models, Genetic ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; Sequence Homology, Amino Acid ; Substrate Specificity ; }, abstract = {In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.}, }
@article {pmid11201749, year = {2001}, author = {Dohoney, KM and Gelles, J}, title = {Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules.}, journal = {Nature}, volume = {409}, number = {6818}, pages = {370-374}, doi = {10.1038/35053124}, pmid = {11201749}, issn = {0028-0836}, mesh = {Adenosine Triphosphatases/metabolism ; Biological Transport ; DNA Helicases/*metabolism ; DNA, Bacterial/*metabolism ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Microscopy ; Microspheres ; Polystyrenes ; }, abstract = {Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.}, }
@article {pmid11163978, year = {2000}, author = {Uno, R and Nakayama, Y and Arakawa, K and Tomita, M}, title = {The orientation bias of Chi sequences is a general tendency of G-rich oligomers.}, journal = {Gene}, volume = {259}, number = {1-2}, pages = {207-215}, doi = {10.1016/s0378-1119(00)00430-3}, pmid = {11163978}, issn = {0378-1119}, mesh = {Bacillus subtilis/genetics ; Base Composition ; Base Sequence ; DNA Replication ; DNA, Bacterial/chemistry/*genetics ; Databases, Factual ; Escherichia coli/genetics ; Genome, Bacterial ; Guanine ; Haemophilus influenzae/genetics ; *Recombination, Genetic ; }, abstract = {The Chi sequences are specific oligomers that stimulate DNA repair by homologous recombination, and are different sequences in each organism. Approximately 75% of the copies of the Chi sequence (5'-GCTGGTGG-3') of Escherichia coli reside on the leading strand, and this orientation bias is often believed to be a consequence of the biological role of Chi sequences as the signal sequence of RecBCD pathway in DNA replication. However, our computer analysis found that many G-rich oligomers also show this asymmetric orientation pattern. The shift in the Chi orientation bias appears around the replication origin and terminus, but these locations are also coincident with the shift points in GC content or GC skew. We conducted the same analysis with the genome of Bacillus subtilis, and found that in addition to Chi, other G-rich oligomers show similar asymmetric orientation patterns, whose shift points were coincident with those of the GC skew. However, the genome of Haemophilus influenzae Rd, whose GC skew is not so pronounced, does not clearly show asymmetric orientation patterns of Chi or other G-rich oligomers. These results lead us to suggest that the uneven distribution of the Chi orientation between the two strands of the double helix is mostly due to the uneven distribution of G content (GC skew) and that the replication-related function of Chi sequences is not the primary factor responsible for the evolutionary pressure causing the orientation bias.}, }
@article {pmid10886371, year = {2000}, author = {El Karoui, M and Schaeffer, M and Biaudet, V and Bolotin, A and Sorokin, A and Gruss, A}, title = {Orientation specificity of the Lactococcus lactis Chi site.}, journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms}, volume = {5}, number = {6}, pages = {453-461}, doi = {10.1046/j.1365-2443.2000.00342.x}, pmid = {10886371}, issn = {1356-9597}, mesh = {Chromosome Inversion ; Chromosomes, Bacterial/genetics ; DNA Replication ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/*metabolism ; Gene Frequency ; Lactococcus lactis/*genetics/metabolism ; Recombination, Genetic ; Regulatory Sequences, Nucleic Acid/*genetics ; }, abstract = {BACKGROUND: In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double-stranded (ds) DNA exonuclease/helicase. Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. ChiEc is frequent and over-represented on its genome, which is thought to be related to its role in dsDNA break repair. We previously identified a Chi-like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram-positive bacterium Lactococcus lactis. ChiLl and RexAB are functional analogues of ChiEc and RecBCD.<br><br>RESULTS: We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. Analysis of ChiLl distribution on the L. lactis chromosome reveals that ChiLl is frequent, highly over-represented, and oriented with respect to the direction of replication.<br><br>CONCLUSION: Our results show that a single orientation of ChiLl interacts with RexAB. The active orientation is preferentially found on the replication leading strand of the L. lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork. We propose that orientation-dependence of Chi activity and over-representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase-Chi couples. Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.}, }
@article {pmid10884344, year = {2000}, author = {Arnold, DA and Handa, N and Kobayashi, I and Kowalczykowski, SC}, title = {A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi.}, journal = {Journal of molecular biology}, volume = {300}, number = {3}, pages = {469-479}, doi = {10.1006/jmbi.2000.3861}, pmid = {10884344}, issn = {0022-2836}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Alleles ; Amino Acid Sequence ; Base Pairing/genetics ; Base Sequence ; DNA, Bacterial/genetics/metabolism ; DNA, Single-Stranded/genetics/metabolism ; Enzyme Activation ; Escherichia coli/*enzymology/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/chemistry/genetics/*metabolism ; Genetic Variation/genetics ; Molecular Sequence Data ; Mutation/*genetics ; Nucleotides/genetics ; Plasmids/genetics/metabolism ; Rec A Recombinases/metabolism ; Recombination, Genetic/*genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; Substrate Specificity ; }, abstract = {In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.}, }
@article {pmid10840065, year = {2000}, author = {Amundsen, SK and Taylor, AF and Smith, GR}, title = {The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {97}, number = {13}, pages = {7399-7404}, pmid = {10840065}, issn = {0027-8424}, support = {R01 GM031693/GM/NIGMS NIH HHS/United States ; R56 GM031693/GM/NIGMS NIH HHS/United States ; GM31693/GM/NIGMS NIH HHS/United States ; }, mesh = {*DNA Repair ; DNA, Bacterial/genetics ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Rec A Recombinases/*genetics ; *Recombination, Genetic ; }, abstract = {The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5'-GCTGGTGG-3'). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. &amp; Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. &amp; Hanawalt, P. (Liss, New York), pp. 413-422; Churchill, J. J., Anderson, D. G. &amp; Kowalczykowski, S. C. (1999) Genes Dev. 13, 901-911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recB(D1080A)CD. We report here that the resulting strain, recB(D1080A)C, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecB(D1080A)C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB(D1080A)CD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi.}, }
@article {pmid10766864, year = {2000}, author = {Arnold, DA and Kowalczykowski, SC}, title = {Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme.}, journal = {The Journal of biological chemistry}, volume = {275}, number = {16}, pages = {12261-12265}, doi = {10.1074/jbc.275.16.12261}, pmid = {10766864}, issn = {0021-9258}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA, Single-Stranded/metabolism ; Electrophoresis, Polyacrylamide Gel ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/metabolism ; Models, Chemical ; Molecular Sequence Data ; Mutagenesis ; Phenotype ; Rec A Recombinases/*metabolism ; *Recombination, Genetic ; }, abstract = {Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme. However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.}, }
@article {pmid10756102, year = {2000}, author = {Chédin, F and Ehrlich, SD and Kowalczykowski, SC}, title = {The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro.}, journal = {Journal of molecular biology}, volume = {298}, number = {1}, pages = {7-20}, doi = {10.1006/jmbi.2000.3556}, pmid = {10756102}, issn = {0022-2836}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Attachment Sites, Microbiological/*genetics ; Bacillus subtilis/*enzymology/*genetics ; Base Sequence ; DNA Helicases/chemistry/*metabolism ; DNA, Bacterial/chemistry/genetics/metabolism ; DNA-Binding Proteins/metabolism ; Evolution, Molecular ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/chemistry/*metabolism ; Exonucleases/chemistry/*metabolism ; Magnesium/metabolism ; Molecular Weight ; Plasmids/chemistry/genetics/metabolism ; Recombination, Genetic/*genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; Sequence Homology, Amino Acid ; Substrate Specificity ; }, abstract = {The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.}, }
@article {pmid10735865, year = {2000}, author = {Handa, N and Ichige, A and Kusano, K and Kobayashi, I}, title = {Cellular responses to postsegregational killing by restriction-modification genes.}, journal = {Journal of bacteriology}, volume = {182}, number = {8}, pages = {2218-2229}, pmid = {10735865}, issn = {0021-9193}, mesh = {Chromosomes, Bacterial/metabolism ; Colony Count, Microbial ; DNA Damage ; DNA Repair ; DNA, Bacterial/metabolism ; Deoxyribonuclease EcoRI/*genetics/metabolism ; Escherichia coli/cytology/enzymology/*genetics/*growth &amp; development ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; Models, Genetic ; Rec A Recombinases/metabolism ; SOS Response, Genetics ; Site-Specific DNA-Methyltransferase (Adenine-Specific)/*genetics/metabolism ; }, abstract = {Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.}, }
@article {pmid10731409, year = {2000}, author = {Churchill, JJ and Kowalczykowski, SC}, title = {Identification of the RecA protein-loading domain of RecBCD enzyme.}, journal = {Journal of molecular biology}, volume = {297}, number = {3}, pages = {537-542}, doi = {10.1006/jmbi.2000.3590}, pmid = {10731409}, issn = {0022-2836}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; T32-GM-07377/GM/NIGMS NIH HHS/United States ; }, mesh = {Base Pairing/genetics ; DNA Helicases/chemistry/genetics/*metabolism ; DNA, Single-Stranded/chemistry/genetics/*metabolism ; DNA, Superhelical/chemistry/genetics/metabolism ; DNA-Binding Proteins/chemistry/genetics/metabolism ; Escherichia coli/*enzymology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*chemistry/genetics/*metabolism ; Holoenzymes/chemistry/genetics/metabolism ; Models, Biological ; Protein Binding ; Rec A Recombinases/*metabolism ; Recombination, Genetic/genetics ; Regulatory Sequences, Nucleic Acid/genetics ; Sequence Deletion/genetics ; }, abstract = {Genetic recombination in Escherichia coli is stimulated by the recombination hotspot Chi (chi), a regulatory element that modifies the activities of the RecBCD enzyme and leads to loading of the DNA strand exchange protein, RecA, onto the chi-containing DNA strand. The RecBC enzyme, which lacks the RecD subunit, loads RecA protein constitutively, in a manner that is independent of chi. Using a truncated RecBC enzyme lacking the 30 kDa C-terminal domain of the RecB subunit, we show that this domain is necessary for RecA protein-loading. We propose that this domain harbors a site that interacts with RecA protein, recruiting it to single-stranded DNA during unwinding. This ability of a translocating enzyme to deliver material (RecA protein) to a specific target site (the chi sequence) parallels that of other cellular motor proteins.}, }
@article {pmid10669596, year = {2000}, author = {Murphy, KC}, title = {Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination.}, journal = {Journal of molecular biology}, volume = {296}, number = {2}, pages = {385-401}, doi = {10.1006/jmbi.1999.3486}, pmid = {10669596}, issn = {0022-2836}, support = {GM51609/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphatases/antagonists &amp; inhibitors/metabolism ; Adenosine Triphosphate/metabolism ; Bacteriophage P22/genetics/growth &amp; development/*physiology ; Bacteriophage lambda/enzymology/genetics/growth &amp; development/physiology ; Carrier Proteins/genetics/*metabolism ; Crosses, Genetic ; DNA Damage/genetics/radiation effects ; DNA, Bacterial/genetics/metabolism/radiation effects ; DNA-Binding Proteins/genetics/*metabolism ; Enzyme Activation ; Escherichia coli/cytology/*enzymology/genetics/virology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/antagonists &amp; inhibitors/chemistry/genetics/*metabolism ; Genetic Complementation Test ; Kinetics ; Mutation/genetics ; Plasmids/genetics/metabolism ; Protein Binding ; Radiation Tolerance ; Recombination, Genetic/*genetics ; Regulatory Sequences, Nucleic Acid/genetics/physiology ; Salmonella typhimurium/cytology/enzymology/genetics/virology ; Ultraviolet Rays ; Viral Proteins/genetics/*metabolism ; }, abstract = {Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination. Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not. In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand. In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity. These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway. This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently.}, }
@article {pmid10617645, year = {2000}, author = {Wang, J and Chen, R and Julin, DA}, title = {A single nuclease active site of the Escherichia coli RecBCD enzyme catalyzes single-stranded DNA degradation in both directions.}, journal = {The Journal of biological chemistry}, volume = {275}, number = {1}, pages = {507-513}, doi = {10.1074/jbc.275.1.507}, pmid = {10617645}, issn = {0021-9258}, support = {GM39777/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; Catalytic Domain ; DNA Helicases/genetics/*metabolism ; DNA, Bacterial/*metabolism ; DNA, Single-Stranded/*metabolism ; Escherichia coli/enzymology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; Models, Biological ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Nucleic Acid Conformation ; Sequence Homology, Amino Acid ; }, abstract = {The RecBCD enzyme of Escherichia coli is an ATP-dependent DNA exonuclease and a helicase. Its exonuclease activity is subject to regulation by an octameric nucleotide sequence called chi. In this study, site-directed mutations were made in the carboxyl-terminal nuclease domain of the RecB subunit, and their effects on RecBCD's enzymatic activities were investigated. Mutation of two amino acid residues, Asp(1067) and Lys(1082), abolished nuclease activity on both single- and double-stranded DNA. Together with Asp(1080), these residues compose a motif that is similar to one shown to form the active site of several restriction endonucleases. The nuclease reactions catalyzed by the RecBCD enzyme should therefore follow the same mechanism as these restriction endonucleases. Furthermore, the mutant enzymes were unable to produce chi-specific fragments that are thought to result from the 3'-5' and 5'-3' single-stranded exonuclease activities of the enzyme during its reaction with chi-containing double-stranded DNA. The results show that the nuclease active site in the RecB C-terminal 30-kDa domain is the universal nuclease active site of RecBCD that is responsible for DNA degradation in both directions during the reaction with double-stranded DNA. A novel explanation for the observed nuclease polarity switch and RecBCD-DNA interaction is offered.}, }
@article {pmid10480929, year = {1999}, author = {Anderson, DG and Churchill, JJ and Kowalczykowski, SC}, title = {A single mutation, RecB(D1080A,) eliminates RecA protein loading but not Chi recognition by RecBCD enzyme.}, journal = {The Journal of biological chemistry}, volume = {274}, number = {38}, pages = {27139-27144}, doi = {10.1074/jbc.274.38.27139}, pmid = {10480929}, issn = {0021-9258}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Helicases/genetics/*metabolism ; DNA Polymerase III/genetics/*metabolism ; DNA, Single-Stranded/metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; Mutagenesis, Site-Directed ; Mutation ; Nucleic Acid Conformation ; Point Mutation ; Rec A Recombinases/*metabolism ; Structure-Activity Relationship ; }, abstract = {Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (chi: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 --> Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether chi sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to chi sites by inactivating in a chi-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.}, }
@article {pmid10197989, year = {1999}, author = {Churchill, JJ and Anderson, DG and Kowalczykowski, SC}, title = {The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi, resulting in constitutive recombination activation.}, journal = {Genes &amp; development}, volume = {13}, number = {7}, pages = {901-911}, pmid = {10197989}, issn = {0890-9369}, support = {R01 GM041347/GM/NIGMS NIH HHS/United States ; T32 GM007377/GM/NIGMS NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; T32-GM-07377/GM/NIGMS NIH HHS/United States ; }, mesh = {Blotting, Southern ; DNA Repair ; DNA, Single-Stranded/*metabolism ; Escherichia coli/genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Genes, Bacterial ; Models, Genetic ; Oligonucleotide Probes ; Rec A Recombinases/*metabolism ; *Recombination, Genetic ; Time Factors ; }, abstract = {Double-strand DNA break repair and homologous recombination in Escherichia coli proceed by the RecBCD pathway, which is regulated by cis-acting elements known as chi sites. A crucial feature of this regulation is the RecBCD enzyme-directed loading of RecA protein specifically onto the 3'-terminal, chi-containing DNA strand. Here we show that RecBC enzyme (lacking the RecD subunit) loads RecA protein constitutively onto the 3'-terminal DNA strand, with no requirement for chi. This strand is preferentially utilized in homologous pairing reactions. We propose that RecA protein loading is a latent property of the RecBCD holoenzyme, which is normally blocked by the RecD subunit and is revealed following interaction with chi.}, }
@article {pmid10197988, year = {1999}, author = {Taylor, AF and Smith, GR}, title = {Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits.}, journal = {Genes &amp; development}, volume = {13}, number = {7}, pages = {890-900}, pmid = {10197988}, issn = {0890-9369}, support = {GM31693/GM/NIGMS NIH HHS/United States ; R01 GM031693/GM/NIGMS NIH HHS/United States ; R56 GM031693/GM/NIGMS NIH HHS/United States ; GM32194/GM/NIGMS NIH HHS/United States ; R01 GM032194/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/metabolism ; Centrifugation, Density Gradient ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Exonucleases/metabolism ; *Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Glycerol/metabolism ; Magnesium/pharmacology ; Models, Biological ; Recombination, Genetic/*physiology ; Sodium Chloride/chemistry ; Time Factors ; }, abstract = {We report here an unusual mechanism for enzyme regulation: the disassembly of all three subunits of RecBCD enzyme after its interaction with a Chi recombination hot spot. The enzyme, which is essential for the major pathway of recombination in Escherichia coli, acts on linear double-stranded DNA bearing a Chi site to produce single-stranded DNA substrates for strand exchange by RecA protein. We show that after reaction with DNA bearing Chi sites, RecBCD enzyme is inactivated and the three subunits migrate as separate species during glycerol gradient ultracentrifugation or native gel electrophoresis. This Chi-mediated inactivation and disassembly of purified RecBCD enzyme can account for the previously reported Chi-dependent loss of Chi activity in E. coli cells containing broken DNA. Our results support a model of recombination in which Chi regulates one RecBCD enzyme molecule to make a single recombinational exchange ('one enzyme-one exchange' hypothesis).}, }
@article {pmid9973617, year = {1999}, author = {El Karoui, M and Amundsen, SK and Dabert, P and Gruss, A}, title = {Gene replacement with linear DNA in electroporated wild-type Escherichia coli.}, journal = {Nucleic acids research}, volume = {27}, number = {5}, pages = {1296-1299}, doi = {10.1093/nar/27.5.1296}, pmid = {9973617}, issn = {0305-1048}, support = {GM31693/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage T4/genetics ; Chromosomes, Bacterial ; DNA, Recombinant/*genetics ; Electroporation ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; Myoviridae/genetics ; Plasmids ; *Recombination, Genetic ; }, abstract = {Gene replacement using linear double-stranded DNA fragments in wild-type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA. Recombination-proficient strains, in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients to overcome this difficulty. Here we report that gene replacements using linear double-stranded donor DNA can be achieved in wild-type E.coli if electrocompetent cells are used. Using a plasmid target, we obtained 10(2)-10(3) gene replacement events/microgram linear DNA. Using an independent chromosomal target, approximately 60 gene replacement events/microgram linear DNA were obtained. The presence of Chi sites on the linear DNA, which are known to block DNA degradation and stimulate recombination in E.coli, had no effect on gene replacement efficiency in either case. RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells. Electrotransformation thus provides a simple way to perform gene replacements in many E.coli strains.}, }
@article {pmid9875851, year = {1998}, author = {Anderson, DG and Kowalczykowski, SC}, title = {Reconstitution of an SOS response pathway: derepression of transcription in response to DNA breaks.}, journal = {Cell}, volume = {95}, number = {7}, pages = {975-979}, doi = {10.1016/s0092-8674(00)81721-3}, pmid = {9875851}, issn = {0092-8674}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacterial Proteins/genetics/metabolism ; Base Sequence ; Chromosome Breakage/physiology ; DNA Damage/*physiology ; DNA Repair/genetics ; DNA-Binding Proteins/metabolism ; Deoxyribonucleases, Type II Site-Specific/metabolism ; Escherichia coli/enzymology/*genetics/metabolism ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; *Gene Expression Regulation, Bacterial/drug effects ; Models, Genetic ; Molecular Sequence Data ; Promoter Regions, Genetic/genetics ; Rec A Recombinases/genetics/metabolism ; Recombination, Genetic/genetics ; Repressor Proteins/genetics/metabolism ; SOS Response, Genetics/*genetics ; Serine Endopeptidases/genetics/metabolism ; Transcription, Genetic/*genetics ; }, abstract = {E. coli responds to DNA damage by derepressing the transcription of about 20 genes that make up the SOS pathway. Genetic analyses have shown that SOS induction in response to double-stranded DNA (dsDNA) breaks requires LexA repressor, and the RecA and RecBCD enzymes--proteins best known for their role as initiators of dsDNA break repair and homologous recombination. Here we demonstrate that purified RecA protein, RecBCD enzyme, single-stranded DNA-binding (SSB) protein, and LexA repressor respond to dsDNA breaks in vitro by derepressing transcription from an SOS promoter. Interestingly, derepression is more rapid if the DNA containing the dsDNA break has a chi recombination hot spot (5'-GCTGGTGG-3'), suggesting a novel regulatory role for one of the most overrepresented octamers in the E. coli genome.}, }
@article {pmid9791113, year = {1998}, author = {Thoms, B and Wackernagel, W}, title = {Interaction of RecBCD enzyme with DNA at double-strand breaks produced in UV-irradiated Escherichia coli: requirement for DNA end processing.}, journal = {Journal of bacteriology}, volume = {180}, number = {21}, pages = {5639-5645}, pmid = {9791113}, issn = {0021-9193}, mesh = {Bacteriophage T4/physiology ; *DNA Damage ; DNA Repair ; DNA, Bacterial/*metabolism/radiation effects ; DNA-Binding Proteins/metabolism ; Escherichia coli/*genetics/radiation effects ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Gene Expression Regulation, Bacterial ; SOS Response, Genetics ; Ultraviolet Rays ; }, abstract = {The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m2). The activity decrease was seen by an increase in survival of phage T4 2(-) of about 200-fold (phage T4 2(-) has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb+) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.}, }
@article {pmid9790841, year = {1998}, author = {Yu, M and Souaya, J and Julin, DA}, title = {Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme.}, journal = {Journal of molecular biology}, volume = {283}, number = {4}, pages = {797-808}, doi = {10.1006/jmbi.1998.2127}, pmid = {9790841}, issn = {0022-2836}, support = {GM39777/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; Bacterial Proteins/chemistry ; Bacteriophage M13/chemistry ; DNA Helicases/metabolism ; DNA, Single-Stranded/metabolism ; DNA, Viral/metabolism ; DNA-Binding Proteins/chemistry ; Endodeoxyribonucleases/*chemistry ; Escherichia coli/*enzymology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*chemistry ; Molecular Sequence Data ; Recombinant Fusion Proteins/*chemistry ; Sequence Alignment ; Substrate Specificity ; Viral Proteins/chemistry ; }, abstract = {The recombinational hot spot chi modulates the nuclease and helicase activities of the RecBCD enzyme, leading to generation of an early DNA intermediate for homologous recombination. Here we identify the subunit location of the nuclease active site in RecBCD. The isolated RecB protein cleaves circular single-stranded M13 phage DNA, but RecB1-929, comprising only the 100 kDa N-terminal domain of RecB, does not. We reported previously that the reconstituted RecB1-929CD enzyme also is not a nuclease, suggesting that the C-terminal 30 kDa domain of RecB is a non-specific ssDNA endonuclease. However, we were unable to detect nuclease activity with the subtilisin-generated C-terminal 30 kDa fragment of RecB. Since the subtilisin-generated fragment did not bind to a ssDNA-agarose column, we designed a chimeric enzyme by attaching the C-terminal 30 kDa domain of RecB to the gene 32 protein of T4 phage, a ssDNA binding protein that does not have strand scission ability. In addition, Asp427 in the chimeric enzyme (Asp1080 in RecB), a residue that is conserved among several RecB homologs, was substituted to alanine (the D427A mutant). The wild-type chimeric enzyme cleaves the M13 DNA and the D427A mutation abolishes the endonuclease activity of the chimeric enzyme but does not affect its DNA binding ability. This finding indicates an unusual bipartite nature in the structural organization of RecB, in which the DNA-binding function is located in the N-terminal 100 kDa domain and the nuclease catalytic domain is located in the C-terminal 30 kDa domain. The purified RecBD1080ACD mutant is a processive helicase but not a nuclease, demonstrating that RecBCD has a single nuclease active site in the C-terminal 30 kDa domain of RecB.}, }
@article {pmid9781875, year = {1998}, author = {Chédin, F and Noirot, P and Biaudet, V and Ehrlich, SD}, title = {A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis.}, journal = {Molecular microbiology}, volume = {29}, number = {6}, pages = {1369-1377}, doi = {10.1046/j.1365-2958.1998.01018.x}, pmid = {9781875}, issn = {0950-382X}, mesh = {Bacillus subtilis/*genetics/*metabolism ; Bacterial Proteins/*genetics/*metabolism ; Base Sequence ; DNA Helicases/*genetics/*metabolism ; DNA Replication/genetics ; DNA, Bacterial/*genetics/*metabolism ; Down-Regulation ; Escherichia coli/enzymology/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/*metabolism ; Genes, Bacterial ; Genome, Bacterial ; Nucleotidyltransferases/*genetics/*metabolism ; Recombination, Genetic ; }, abstract = {Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.}, }
@article {pmid9735287, year = {1998}, author = {Anderson, DG and Kowalczykowski, SC}, title = {SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates.}, journal = {Journal of molecular biology}, volume = {282}, number = {2}, pages = {275-285}, doi = {10.1006/jmbi.1998.2013}, pmid = {9735287}, issn = {0022-2836}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Helicases/chemistry/metabolism/physiology ; DNA, Bacterial/metabolism ; DNA, Single-Stranded/metabolism/physiology ; DNA-Binding Proteins/chemistry/metabolism/*physiology ; Escherichia coli/enzymology ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/chemistry/metabolism/*physiology ; Models, Biological ; }, abstract = {The RecBCD enzyme of Escherichia coli initiates homologous recombination by unwinding and simultaneously degrading DNA from a double-stranded DNA end. Single-stranded DNA loops are intermediates of this unwinding process. Here we show that SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding, by binding to these ssDNA intermediates. Prior to interaction with the recombination hot spot chi, RecBCD enzyme has both 3'-->5' exonuclease and a weaker 5'-->3' exonuclease activity. We show that degradation of the 5'-terminal strand at the entry site is much more extensive in the absence of SSB protein. After interaction with chi, the level of 5'-->3' exonuclease activity is increased; as expected, degradation of the 5'-strand is also elevated in the absence of SSB protein. Furthermore, we show that, in the absence of SSB protein, the RecBCD enzyme is inhibited by the ssDNA products of unwinding; SSB protein alleviates this inhibition. These results provide insight into the organization of helicase and nuclease domains within the RecBCD enzyme, and also suggest a new level at which the nuclease activity of RecBCD enzyme is controlled. Hence, they offer new insight into the role of SSB protein in the initiation phase of recombination.}, }
@article {pmid9632715, year = {1998}, author = {Arnold, DA and Bianco, PR and Kowalczykowski, SC}, title = {The reduced levels of chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo.}, journal = {The Journal of biological chemistry}, volume = {273}, number = {26}, pages = {16476-16486}, doi = {10.1074/jbc.273.26.16476}, pmid = {9632715}, issn = {0021-9258}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphatases/metabolism ; DNA Helicases/metabolism ; DNA, Bacterial/*metabolism ; DNA, Single-Stranded/metabolism ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; Exonucleases/metabolism ; Mutation ; Nucleotides/*metabolism ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; Structure-Activity Relationship ; }, abstract = {Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme and is stimulated by an 8-nucleotide element known as Chi (chi). We present a detailed biochemical characterization of a mutant RecBCD enzyme, designated RecBC1004D, that displays a reduced level of chi site recognition. Initially characterized genetically as unable to respond to the chi sequence, we provide evidence to indicate that the ability of this mutant enzyme to respond to chi is reduced rather than lost; the mutant displays about 20-fold lower chi recognition than wild-type RecBCD enzyme. Although this enzyme exhibits wild-type levels of double-stranded DNA exonuclease, helicase, and ATPase activity, its ability to degrade single-stranded DNA is enhanced 2-3-fold. The data presented here suggest that the reduced recombination proficiency of the recBC1004D strain observed in vivo results from a basal level of modification of the RecBC1004D enzyme at both chi-specific, as well as nonspecific, DNA sequences.}, }
@article {pmid9547270, year = {1998}, author = {Amundsen, SK and Taylor, AF and Smith, GR}, title = {A stimulatory RNA associated with RecBCD enzyme.}, journal = {Nucleic acids research}, volume = {26}, number = {9}, pages = {2125-2131}, pmid = {9547270}, issn = {0305-1048}, support = {GM31693/GM/NIGMS NIH HHS/United States ; GM32194/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Helicases/isolation &amp; purification/*metabolism ; DNA, Bacterial/metabolism ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/isolation &amp; purification/*metabolism ; Nucleic Acid Conformation ; RNA, Bacterial/isolation &amp; purification/*metabolism ; *Recombination, Genetic ; Ribonucleoproteins/isolation &amp; purification/*metabolism ; Sequence Analysis, RNA ; }, abstract = {RecBCD enzyme acts in the major pathway of homologous recombination of linear DNA in Escherichia coli. The enzyme unwinds DNA and is an ATP-dependent double-strand and single-strand exonuclease and a single-strand endonuclease; it acts at Chi recombination hotspots (5'-GCTGGTGG-3') to produce a recombinogenic single-stranded DNA 3'-end. We found that a small RNA with a unique sequence of approximately 24 nt was tightly bound to RecBCD enzyme and co-purified with it. When added to native enzyme this RNA, but not four others, increased DNA unwinding and Chi nicking activities of the enzyme. In seven similarly active enzyme preparations the molar ratio of RNA molecules to RecBCD enzyme molecules ranged from 0.2 to <0.008. These results suggest that, although this unique RNA is not an essential enzyme subunit, it has a biological role in stimulating RecBCD enzyme activity.}, }
@article {pmid9535091, year = {1998}, author = {Sourice, S and Biaudet, V and El Karoui, M and Ehrlich, SD and Gruss, A}, title = {Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site.}, journal = {Molecular microbiology}, volume = {27}, number = {5}, pages = {1021-1029}, doi = {10.1046/j.1365-2958.1998.00749.x}, pmid = {9535091}, issn = {0950-382X}, mesh = {Base Sequence ; Colony Count, Microbial ; Consensus Sequence ; DNA Helicases/genetics/*metabolism ; DNA, Bacterial/*metabolism ; Escherichia coli/*genetics/metabolism ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; Genome, Bacterial ; Haemophilus influenzae/*genetics/metabolism ; Molecular Weight ; Oligonucleotides/biosynthesis ; Plasmids ; Recombination, Genetic ; Regulatory Sequences, Nucleic Acid/*genetics ; Sequence Analysis ; Substrate Specificity ; Transformation, Bacterial ; }, abstract = {The Escherichia coli Chi site 5'-GCTGGTGG-3' modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E. coli genome. Chi is also present much more frequently than predicted statistically for a random 8-mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae. The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5'-GNTGGTGG-3' and 5'-G(G/C)TGGAGG-3', showed Chi activity. As in E. coli, the H. influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over-represented, this feature is less marked than that of the E. coli Chi site. In contrast to E. coli, the H. influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.}, }
@article {pmid9504905, year = {1998}, author = {Friedman-Ohana, R and Karunker, I and Cohen, A}, title = {Chi-dependent intramolecular recombination in Escherichia coli.}, journal = {Genetics}, volume = {148}, number = {2}, pages = {545-557}, pmid = {9504905}, issn = {0016-6731}, mesh = {Bacteriophages/chemistry ; DNA, Viral/metabolism ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; Genotype ; Mutation/genetics ; Oligodeoxyribonucleotides/*metabolism ; Rec A Recombinases/metabolism ; Recombination, Genetic/*genetics ; }, abstract = {Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5'-GCTGGTGG-3') that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.}, }
@article {pmid9448271, year = {1998}, author = {Yu, M and Souaya, J and Julin, DA}, title = {The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {95}, number = {3}, pages = {981-986}, pmid = {9448271}, issn = {0027-8424}, support = {GM39777/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Helicases/*chemistry/genetics/*metabolism ; DNA, Bacterial/metabolism ; Endodeoxyribonucleases/*chemistry/genetics/*metabolism ; Escherichia coli/enzymology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*chemistry/genetics/*metabolism ; Molecular Weight ; Mutagenesis, Site-Directed ; Sequence Deletion ; Structure-Activity Relationship ; }, abstract = {The RecBCD enzyme from Escherichia coli is an ATP-dependent helicase and an ATP-stimulated nuclease. The 3' --> 5' exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a recombinational hot spot, called chi (chi). We have prepared a RecB deletion mutant (RecB1-929) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains. The RecB1-929 protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase. Reconstitution of RecB1-929 with RecC and RecD leads to processive unwinding of a linearized plasmid. However, the reconstituted RecB1-929CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme. These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD. On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD's transformation from a 3' --> 5' exonuclease to a helicase when it meets a chi site.}, }
@article {pmid9382825, year = {1997}, author = {Eggleston, AK and West, SC}, title = {Recombination initiation: easy as A, B, C, D... chi?.}, journal = {Current biology : CB}, volume = {7}, number = {12}, pages = {R745-9}, doi = {10.1016/s0960-9822(06)00394-0}, pmid = {9382825}, issn = {0960-9822}, mesh = {Binding Sites ; DNA, Bacterial/*genetics ; Escherichia coli/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Mutation ; Rec A Recombinases/*metabolism ; *Recombination, Genetic ; }, abstract = {The octameric Chi (chi) sequence is a recombination hotspot in Escherichia coli. Recent studies suggest a singular mechanism by which chi regulates not only the nuclease activity of RecBCD enzyme, but also the ability of RecBCD to promote loading of the strand exchange protein, RecA, onto chi-containing DNA.}, }
@article {pmid9383046, year = {1997}, author = {Corre, J and Cornet, F and Patte, J and Louarn, JM}, title = {Unraveling a region-specific hyper-recombination phenomenon: genetic control and modalities of terminal recombination in Escherichia coli.}, journal = {Genetics}, volume = {147}, number = {3}, pages = {979-989}, pmid = {9383046}, issn = {0016-6731}, mesh = {Chromosomes, Bacterial ; DNA Replication ; DNA, Bacterial/genetics/metabolism ; Escherichia coli/*genetics/metabolism ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; *Recombination, Genetic ; Repetitive Sequences, Nucleic Acid ; }, abstract = {The propensity of the terminus of the Escherichia coli chromosome for recombination has been further explored, using a test based on the selectable loss of a lambda prophage inserted between repeated sequences from Tn10. Terminal recombination appears region-specific and unrelated to replication termination in a strain harboring a major chromosomal rearrangement. It requires RecBC(D) activity and must therefore occur between sister chromosomes, to conserve genomic integrity in spite of DNA degradation by RecBCD. Terminal recombination is maximal in the dif region and its intensity on either side of this recombination site depends on the orientation of the repeated sequences, probably because of the single chi site present in each repeat. Additional observations support the model that the crossover is initiated by single-strand invasion between sister chromosomes followed by RecBCD action as a consequence of DNA breakage due to the initial invasion event. Crossover location within repeats inserted at dif position supports the possibility that sister chromosomes are tightly paired in the centre of the terminal recombination zone. These data reinforce the model that postreplicative reconstruction of nucleoid organization creates a localized synapsis between the termini of sister chromosomes.}, }
@article {pmid9435243, year = {1998}, author = {el Karoui, M and Ehrlich, D and Gruss, A}, title = {Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {95}, number = {2}, pages = {626-631}, pmid = {9435243}, issn = {0027-8424}, mesh = {Base Sequence ; Cloning, Molecular ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; *Genes, Bacterial ; Lactococcus lactis/*enzymology ; Molecular Sequence Data ; Plasmids ; *Recombination, Genetic ; }, abstract = {Studies of RecBCD-Chi interactions in Escherichia coli have served as a model to understand recombination events in bacteria. However, the existence of similar interactions has not been demonstrated in bacteria unrelated to E. coli. We developed an in vivo model to examine components of dsDNA break repair in various microorganisms. Here, we identify the major exonuclease in Lactococcus lactis, a Gram-positive organism evolutionarily distant from E. coli, and provide evidence for exonuclease-Chi interactions. Insertional mutants of L. lactis, screened as exonuclease-deficient, affected a single locus and resulted in UV sensitivity and recombination deficiency. The cloned lactococcal genes (called rexAB) restored UV resistance, recombination proficiency, and the capacity to degrade linear DNA, to an E. coli recBCD mutant. In this context, DNA degradation is specifically blocked by the putative lactococcal Chi site (5'-GCGCGTG-3'), but not by the E. coli Chi (5'-GCTGGTGG-3') site. RexAB-mediated recombination was shown to be stimulated approximately 27-fold by lactococcal Chi. Our results reveal that RexAB fulfills the biological roles of RecBCD and indicate that its activity is modulated by a short DNA sequence. We speculate that exonuclease/recombinase enzymes whose activities are modulated by short DNA sequences are widespread among bacteria.}, }
@article {pmid9348042, year = {1997}, author = {Handa, N and Ohashi, S and Kusano, K and Kobayashi, I}, title = {Chi-star, a chi-related 11-mer sequence partially active in an E. coli recC1004 strain.}, journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms}, volume = {2}, number = {8}, pages = {525-536}, doi = {10.1046/j.1365-2443.1997.1410339.x}, pmid = {9348042}, issn = {1356-9597}, mesh = {Bacteriophage lambda/genetics ; Cloning, Molecular ; DNA Replication ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics ; *Mutation ; Plasmids ; Recombination, Genetic ; }, abstract = {BACKGROUND: chi sequence (5'GCTGGTGG) of Escherichia coli was first identified as a site that increased the plaque size of bacteriophage lambda. Subsequent studies showed that this site is responsible for both the attenuation ofRecBCD exonuclease activity and the promotion of RecA, RecBCD-mediated recombination. It is known that bacteriophage lambda containing the chi site makes very small plaques on a recC* (recC1004) mutant because chi is not recognized by the RecBC*D mutant enzyme.<br><br>RESULTS: We cloned E. coli chromosomal fragments in lambda which allowed lambda to form larger plaques on this recC1004 mutant. The fragments were found to share a chi-like 11-mer sequence, 5'GCTGGTGCTCG. Substitution of these fragments with a synthetic 11-mer of this sequence and single-base-pair substitution analysis of its last four nucleotides demonstrated that this sequence is both necessary and sufficient for the observed activity. The sequence, designated X* (chi-star), protected rolling-circle DNA replication in the recC1004 mutant and in the recBCD+ strain, most likely because it attenuated the exonuclease activity of the RecBC*D and RecBCD+ enzyme. chi-star, did not significantly stimulate lambda recombination in two assays.<br><br>CONCLUSION: We have discovered that a mutant RecBCD enzyme responds, in vivo, to a longer chi variant.}, }
@article {pmid9230304, year = {1997}, author = {Anderson, DG and Kowalczykowski, SC}, title = {The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner.}, journal = {Cell}, volume = {90}, number = {1}, pages = {77-86}, doi = {10.1016/s0092-8674(00)80315-3}, pmid = {9230304}, issn = {0092-8674}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Base Sequence ; Binding Sites ; DNA Helicases/metabolism ; DNA Repair ; DNA, Bacterial/*chemistry/genetics/metabolism ; DNA, Single-Stranded/*metabolism ; Escherichia coli/*enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Models, Structural ; Nucleic Acid Conformation ; Plasmids ; Rec A Recombinases/*metabolism ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; Substrate Specificity ; }, abstract = {Double-stranded DNA break repair and homologous recombination in E. coli are initiated by the RecBCD enzyme, which unwinds and simultaneously degrades DNA from a double-stranded DNA end. This process is stimulated by cis-acting DNA elements, known as chi sites. Using both in vitro pairing and nuclease protection assays, we demonstrate that the translocating RecBCD enzyme, which has been activated by chi, coordinates the preferential loading of the homologous pairing protein, RecA, onto the resultant single-stranded DNA downstream of chi. This facilitated loading of RecA protein results in a substantial increase in both the efficiency and rate of in vitro recombination reactions and offers an explanation for stimulation of recombination and repair in vivo by chi.}, }
@article {pmid9192629, year = {1997}, author = {Bianco, PR and Kowalczykowski, SC}, title = {The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {94}, number = {13}, pages = {6706-6711}, pmid = {9192629}, issn = {0027-8424}, support = {R01 GM041347/GM/NIGMS NIH HHS/United States ; GM 41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA, Recombinant/genetics ; DNA, Single-Stranded/*genetics ; Escherichia coli/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid/genetics ; }, abstract = {The RecBCD enzyme of Escherichia coli functions in the seemingly disparate roles of homologous recombination and the degradation of DNA. Which of these two roles it assumes is regulated by the 8-base recombination hotspot, Chi. Using double-stranded DNA substrates that are heteroduplex at the Chi locus we have established the determinants for Chi recognition. Our results show that an actively translocating RecBCD enzyme requires only the sequence information in the 5'-GCTGGTGG-3'-containing strand to recognize and to be regulated by Chi. Furthermore, the RecBCD enzyme can translocate through DNA heteroduplex bubbles as large as 22 bases, and still recognize a Chi sequence embedded in this region. This implies that recognition of Chi occurs following the unwinding of the DNA.}, }
@article {pmid9092551, year = {1997}, author = {Chen, HW and Ruan, B and Yu, M and Wang, Jd and Julin, DA}, title = {The RecD subunit of the RecBCD enzyme from Escherichia coli is a single-stranded DNA-dependent ATPase.}, journal = {The Journal of biological chemistry}, volume = {272}, number = {15}, pages = {10072-10079}, doi = {10.1074/jbc.272.15.10072}, pmid = {9092551}, issn = {0021-9258}, support = {GM39777/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphatases/*metabolism ; Adenosine Triphosphate/*metabolism ; Chromatography, Affinity ; DNA Helicases/*chemistry ; DNA, Single-Stranded/metabolism ; Electrophoresis, Polyacrylamide Gel ; Endodeoxyribonucleases/*chemistry ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*chemistry ; *Histidine ; Hydrolysis ; Protein Conformation ; }, abstract = {We have expressed the RecD subunit of the RecBCD enzyme from Escherichia coli as a fusion protein with a 31-amino acid NH2-terminal extension including 6 consecutive histidine residues (HisRecD). The overexpressed fusion protein can be purified in urea-denatured form by metal chelate affinity chromatography. The mixture of renatured HisRecD protein and the RecB and RecC proteins has a high level of ATP-dependent nuclease activity with either single- or double-stranded DNA, enhanced DNA unwinding activity, enhanced ATP hydrolysis activity in the presence of a small DNA oligomer cosubstrate, and chi-cutting activity. These are all characteristics of the RecBCD holoenzyme. The HisRecD protein by itself hydrolyzes ATP in the presence of high concentrations of single-stranded DNA (polydeoxythymidine). The activity is unstable at 37 degrees C, but is measurable at room temperature (about 23 degrees C). The HisRecD has very little ATPase activity in the presence of a much shorter single-stranded DNA (oligodeoxy(thymidine)12). HisRecD hydrolyzes ATP more efficiently than GTP and UTP, and has very little activity with CTP. We also purified a fusion protein containing a Lys to Gln mutation in the putative ATP-binding site of RecD. This mutant protein has no ATPase activity, indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.}, }
@article {pmid9093843, year = {1997}, author = {Dabert, P and Smith, GR}, title = {Gene replacement with linear DNA fragments in wild-type Escherichia coli: enhancement by Chi sites.}, journal = {Genetics}, volume = {145}, number = {4}, pages = {877-889}, pmid = {9093843}, issn = {0016-6731}, support = {GM-31693/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacterial Proteins/metabolism ; DNA, Bacterial/*genetics ; DNA, Recombinant/*genetics ; DNA, Single-Stranded/genetics ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; *Genes, Bacterial ; Rec A Recombinases/metabolism ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; Species Specificity ; Transduction, Genetic ; *Transformation, Bacterial ; }, abstract = {During conjugation and transduction of Escherichia coli even numbers of recombinational exchanges are required for replacement of a gene on the circular chromosome. We studied gene replacement using a related method of gene transfer (transformation with 6.5-kb linear DNA fragments) as an experimental model for conjugation and transduction. Two properly situated Chi sites, 5' GCTGGTGG 3', stimulated gene replacement approximately 50-fold, more than the sum of the stimulation by the individual Chi sites. Gene replacement was dependent on RecA and RecB functions. Similar results were obtained with an alternative experimental model in which linear DNA fragments were generated from phage lambda by intracellular EcoRI restriction following infection. Dual Chi site-stimulation of these RecA-, RecB-dependent recombination events thus did not depend upon the mode of delivery of the linear DNA into the cells. A single DNA fragment with two Chi sites was sufficient for gene replacement. These results support a one Chi-one exchange hypothesis ("long chunk" gene replacement), stemming from studies with purified RecBCD enzyme, and argue against models in which Chi converts RecBCD enzyme to a state capable of promoting multiple exchanges on one DNA molecule. These results also provide a method for gene targeting in wild-type E. coli and suggest a method for gene targeting in other organisms.}, }
@article {pmid9119222, year = {1997}, author = {Anderson, DG and Kowalczykowski, SC}, title = {The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme.}, journal = {Genes &amp; development}, volume = {11}, number = {5}, pages = {571-581}, doi = {10.1101/gad.11.5.571}, pmid = {9119222}, issn = {0890-9369}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Binding Sites ; DNA/genetics/*metabolism ; DNA, Single-Stranded/genetics/metabolism ; Escherichia coli/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/*metabolism ; *Recombination, Genetic ; *Regulatory Sequences, Nucleic Acid ; Substrate Specificity ; Up-Regulation ; }, abstract = {Homologous recombination in Escherichia coli is stimulated at DNA sequences known as chi sites. Stimulation requires the multifunctional RecBCD enzyme, which is both a helicase and a 3' --> 5' exonuclease. Upon recognition of a properly oriented chi site, the 3' --> 5' exonuclease activity is attenuated. Here we show that in addition to attenuation of the 3' --> 5' exonuclease activity, recognition of chi by the RecBCD enzyme also up-regulates a nuclease activity of the opposite polarity, resulting in an enzyme that now preferentially degrades 5' --> 3'. These results demonstrate that chi is a unique regulatory element that converts the antirecombinogenic form of the RecBCD enzyme into a recombinogenic form by causing two distinct enzymatic changes: attenuation of the 3' --> 5' nuclease activity, and up-regulation of the 5' --> 3' nuclease activity. The consequence of chi recognition is the production of a recombination intermediate possessing a 3'-ssDNA overhang terminating at the chi sequence. This processing of a dsDNA end to a 3'-ssDNA overhang parallels that which occurs during the initation of homologous recombination in other pathways in E. coli, and in other organisms such as the yeast Saccharomyces cerevisiae.}, }
@article {pmid9167969, year = {1997}, author = {Anderson, DG and Churchill, JJ and Kowalczykowski, SC}, title = {Chi-activated RecBCD enzyme possesses 5'--&gt;3' nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit.}, journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms}, volume = {2}, number = {2}, pages = {117-128}, doi = {10.1046/j.1365-2443.1997.1130311.x}, pmid = {9167969}, issn = {1356-9597}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA/metabolism ; DNA Helicases/genetics/*metabolism ; DNA, Single-Stranded/metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Enzyme Activation ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; *Regulatory Sequences, Nucleic Acid ; }, abstract = {BACKGROUND: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (chi) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of chi causes both attenuation of the 3'-->5' exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5'-->3' polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that chi-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit.<br><br>RESULTS: Through examination of RecBCD exonuclease activity under a variety of conditions, we have shown that recognition of chi by the RecBCD enzyme results in a net reduction of nuclease activity. In addition, the exact location of the first cleavage event elicited by chi-activation of the 5'-->3' nuclease is dependent upon the concentration of free magnesium ions. Finally, we have demonstrated that purified RecBC enzyme (i.e. without the RecD subunit) possesses no significant exonuclease activity under conditions where the chi-modified RecBCD enzyme is an active 5'-->3' exonuclease.<br><br>CONCLUSIONS: We have shown that, despite the activation of a 5'-->3' exonuclease, recognition of chi by the RecBCD enzyme results in a net preservation of DNA. This new chi-activated nucleolytic action shows surprising variability in the exact location of its initial cleavage. We have demonstrated that purified RecBC enzyme is not an exact analogue of the chi-activated RecBCD enzyme, suggesting that the biochemical basis of chi-activation is not simply ejection of the RecD subunit.}, }
@article {pmid9006046, year = {1997}, author = {Kuzminov, A and Stahl, FW}, title = {Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation.}, journal = {Journal of bacteriology}, volume = {179}, number = {3}, pages = {880-888}, pmid = {9006046}, issn = {0021-9193}, mesh = {Chromosomes, Bacterial/*metabolism ; Cosmids/*metabolism ; DNA Damage ; DNA Repair ; DNA Replication ; Escherichia coli/*genetics/metabolism ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Models, Genetic ; Mutation ; Rec A Recombinases/*genetics ; Recombination, Genetic ; }, abstract = {To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair. Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease. We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities. Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme. We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells. Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease. A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E. coli is discussed.}, }
@article {pmid9689227, year = {1997}, author = {Handa, N and Ohashi, S and Kobayashi, I}, title = {Clustering of chi sequence in Escherichia coli genome.}, journal = {Microbial &amp; comparative genomics}, volume = {2}, number = {4}, pages = {287-298}, doi = {10.1089/omi.1.1997.2.287}, pmid = {9689227}, issn = {1090-6592}, mesh = {Bacteriophage lambda/physiology ; Base Sequence ; DNA Helicases/genetics ; DNA, Bacterial/*genetics ; *DNA-Binding Proteins ; Escherichia coli/*genetics/virology ; Molecular Sequence Data ; Sequence Alignment ; Trans-Activators/genetics ; }, abstract = {An 8-mer DNA sequence called chi (5'-GCTGGTGG) is present on the Escherichia coli chromosome at a high frequency. It is responsible for both the attenuation of RecBCD exonuclease activity and the promotion of RecABCD-mediated homologous recombination. chi was first identified as a site that increased plaque size of bacteriophage lambda. lambda containing chi makes very small plaques on a recC* (recC1004) mutant because chi is poorly recognized by the RecBC*D mutant enzyme. We cloned E. coli chromosomal fragments in lambda that allowed lambda to form larger plaques on this recC* mutant as well as on the rec+ parent. One identified fragment contained a cluster of two copies of chi and several chi-like sequences with the same orientation. It increased recombination in the rec+ strain more than a fragment with one chi did. This fragment was within the rep gene, whose helicase product is known to be required for growth in the absence of functional RecBCD enzyme. The possibility that RecBCD enzyme might interact both with the rep gene and its product is discussed. Many of the other chi clusters identified in the E. coli genome database lie within genes for membrane proteins. The possible significance of these findings is discussed.}, }
@article {pmid8682788, year = {1996}, author = {Zaman, MM and Boles, TC}, title = {Plasmid recombination by the RecBCD pathway of Escherichia coli.}, journal = {Journal of bacteriology}, volume = {178}, number = {13}, pages = {3840-3845}, pmid = {8682788}, issn = {0021-9193}, mesh = {Base Sequence ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Molecular Sequence Data ; Mutation ; Plasmids/*genetics ; *Recombination, Genetic ; }, abstract = {Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M. M. Zaman and T. C. Boles, J. Bacteriol. 176:5093-5100, 1994). Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A. J. Clark and K. B. Low, p. 155-215, in K. B. Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains. Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains. Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes. The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination. Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells.}, }
@article {pmid8852834, year = {1996}, author = {Razavy, H and Szigety, SK and Rosenberg, SM}, title = {Evidence for both 3' and 5' single-strand DNA ends in intermediates in chi-stimulated recombination in vivo.}, journal = {Genetics}, volume = {142}, number = {2}, pages = {333-339}, pmid = {8852834}, issn = {0016-6731}, mesh = {Base Sequence ; *DNA, Single-Stranded ; Exodeoxyribonucleases/*genetics/metabolism ; Models, Genetic ; Molecular Sequence Data ; Oligodeoxyribonucleotides ; *Recombination, Genetic ; }, abstract = {This paper focuses on elucidation of the structures of intermediates in recombination stimulated by Chi recombination hotspots in vivo. We report that null mutations in genes encoding single-strand exonucleases of 3' polarity, Exonuclease I (Exo I), of 5' polarity, RecJ, and of both polarities, Exo VII, alter the ability of Chi sites to promote recombination, and diminish the frequency of recombination. Maximal effects occur when single-strand exonucleases of both polarities are eliminated. These data imply that 3' and 5' single-strand DNA ends, the substrates for these exonucleases, exist in bona fide, product-generating intermediates in Chi-stimulated recombination in vivo. These results also identify three new proteins not known previously to affect RecBCD-mediated recombination.}, }
@article {pmid8582627, year = {1995}, author = {Myers, RS and Stahl, MM and Stahl, FW}, title = {Chi recombination activity in phage lambda decays as a function of genetic distance.}, journal = {Genetics}, volume = {141}, number = {3}, pages = {805-812}, pmid = {8582627}, issn = {0016-6731}, support = {5F32GM-14196/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacterial Proteins/metabolism ; Bacteriophage lambda/*genetics ; DNA Helicases/metabolism ; DNA Replication ; DNA, Single-Stranded/metabolism ; DNA, Viral/biosynthesis/genetics ; Escherichia coli/enzymology/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; Models, Genetic ; *Recombination, Genetic ; Sequence Homology, Nucleic Acid ; }, abstract = {In Escherichia coli, chi is a recombination hotspot that stimulates RecBCD-dependent exchange at and to one side of itself. chi activity is highest at chi and decreases with distance from chi. The decrease in chi activity may be a simple property of the physical distance over which chi can stimulate recombination. Alternatively, the decay in chi activity with distance may reflect the high likelihood that chi-stimulated recombination occurs in a single chi-proximal act, to the exclusion of additional chi-stimulated exchanges more distal to chi. To test the models, we determined if chi activity decreases as a function of physical distance (i.e., DNA base pairs) or genetic distance (homologous DNA base pairs). Our results indicate that chi activity decays as a function of genetic distance. In addition, we found that the sbcB gene product (exonuclease I, a 3'-->5' ssDNA exonuclease) modulates the distance over which chi can act. In contrast, the recJ gene product (a 5'-->3' ssDNA exonuclease) does not alter the decay of chi activity.}, }
@article {pmid7592661, year = {1995}, author = {Taylor, AF and Smith, GR}, title = {Strand specificity of nicking of DNA at Chi sites by RecBCD enzyme. Modulation by ATP and magnesium levels.}, journal = {The Journal of biological chemistry}, volume = {270}, number = {41}, pages = {24459-24467}, doi = {10.1074/jbc.270.41.24459}, pmid = {7592661}, issn = {0021-9258}, support = {GM31693/GM/NIGMS NIH HHS/United States ; GM32194/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/*pharmacology ; Base Sequence ; Binding Sites ; DNA/chemistry/*metabolism ; DNA Helicases/*metabolism ; Escherichia coli/*enzymology/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Genes, Bacterial ; Magnesium/*pharmacology ; Models, Genetic ; Models, Structural ; Molecular Sequence Data ; Plasmids/*chemistry/*metabolism ; Recombination, Genetic ; Substrate Specificity ; }, abstract = {RecBCD enzyme is essential for the major pathway of homologous recombination of linear DNA in Escherichia coli. It is a potent nuclease and helicase and, during its unwinding of double-stranded DNA, makes single-strand scissions in the vicinity of Chi recombination hot spots. We report here that both the strand that is cut and the position of the cuts relative to Chi depended on the ATP to Mg2+ ratio. With ATP in excess, Chi-dependent nicks occurred, as we have previously reported, four to six nucleotides to the 3'-side of the Chi octamer (5'-GCTGGTGG-3') and were detected only on the strand bearing that sequence. Three differences were seen with Mg2+ in excess. 1) Chi-dependent 3'-ends were produced on the GCTGGTGG-containing strand closer to and within the Chi octamer. 2) Chi-dependent cuts occurred on the complementary DNA strand. 3) RecBCD enzyme destroyed the 3'-terminated strand of DNA from its entry point up to the vicinity of the Chi site, as others have previously reported. We show here that, with Mg2+ in excess, the enzyme continued to travel along DNA, after encountering a Chi site, releasing both strands of the DNA distal to Chi as single strands. We discuss potential biological consequences of these two modes of RecBCD enzyme-Chi interaction.}, }
@article {pmid7560868, year = {1995}, author = {Stahl, F and Myers, R}, title = {Old and new concepts for the role of chi in bacterial recombination.}, journal = {The Journal of heredity}, volume = {86}, number = {5}, pages = {327-329}, doi = {10.1093/oxfordjournals.jhered.a111599}, pmid = {7560868}, issn = {0022-1503}, mesh = {Base Sequence ; DNA Helicases/metabolism ; DNA, Bacterial/chemistry/*genetics ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; Mutation ; *Recombination, Genetic ; }, abstract = {The DNA sequence 5'[GCTGGTGG]3', which is called chi, stimulates recombination that is mediated by the RecBCD pathway of Escherichia coli. In 1981, a model was proposed in which the RecBCD enzyme enters DNA at a double-chain end. The enzyme then travels between the chains by unwinding and rewinding the DNA at different rates so that the traveling enzyme becomes encumbered by a region of unwound DNA. Upon meeting chi, the enzyme was supposed to cut one of the two unwound chains, generating thereby a recombinagenic single-chain end. The model, based on microscopical observations of RecBCD enzyme interacting with linear duplex DNA, was supported by the subsequent finding that RecBCD acting in vitro under certain conditions did deliver a nick at chi. This widely embraced model has been challenged by a model in which the exonuclease activity of RecBCD destroys DNA from the enzyme's entry site to chi. The role of chi according to the new model is to inhibit this nuclease activity of RecBCD, perhaps by ejecting the RecD subunit from the enzyme, thereby revealing the enzyme's recombinase activity.}, }
@article {pmid7608206, year = {1995}, author = {Dixon, DA and Kowalczykowski, SC}, title = {Role of the Escherichia coli recombination hotspot, chi, in RecABCD-dependent homologous pairing.}, journal = {The Journal of biological chemistry}, volume = {270}, number = {27}, pages = {16360-16370}, doi = {10.1074/jbc.270.27.16360}, pmid = {7608206}, issn = {0021-9258}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Helicases/*metabolism ; DNA, Bacterial/genetics ; DNA, Single-Stranded/metabolism ; DNA-Binding Proteins/metabolism ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; Magnesium/pharmacology ; Models, Genetic ; Mutation ; Nucleic Acid Conformation/drug effects ; Rec A Recombinases/*metabolism ; Recombination, Genetic/*genetics ; }, abstract = {Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as chi sites, 5'-GCTGGTGG-3'. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a chi sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of chi-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and chi-specific single-stranded DNA fragment production are observed. Also, under these conditions, chi-specific fragments derived from both the upstream and downstream regions of the DNA strand containing chi and from cleavage of the non-chi-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD not equal to) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to chi stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, chi itself may be a preferred site for initiation of homologous pairing in this concerted process.}, }
@article {pmid7603978, year = {1995}, author = {Myers, RS and Kuzminov, A and Stahl, FW}, title = {The recombination hot spot chi activates RecBCD recombination by converting Escherichia coli to a recD mutant phenocopy.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {92}, number = {14}, pages = {6244-6248}, pmid = {7603978}, issn = {0027-8424}, support = {5F32GM14196/GM/NIGMS NIH HHS/United States ; GM33677/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/genetics ; Base Sequence ; Chromosomes, Bacterial ; Cosmids ; DNA Replication ; DNA, Bacterial/genetics ; DNA, Viral/genetics/metabolism ; Escherichia coli/enzymology/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*biosynthesis/*genetics ; *Genes, Bacterial ; Genotype ; Molecular Sequence Data ; Mutagenesis ; Plasmids ; *Recombination, Genetic ; Restriction Mapping ; }, abstract = {The products of the recB and recC genes are necessary for conjugal recombination and for repair of chromosomal double-chain breaks in Escherichia coli. The recD gene product combines with the RecB and RecC proteins to comprise RecBCD enzyme but is required for neither recombination nor repair. On the contrary, RecBCD enzyme is an exonuclease that inhibits recombination by destroying linear DNA. The RecD ejection model proposes that RecBCD enzyme enters a DNA duplex at a double-chain end and travels destructively until it encounters the recombination hot spot sequence chi. Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer. With the loss of the RecD subunit, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase. To test the model, genetic crosses between lambda phage were conducted in cells containing chi on a nonhomologous plasmid. Upon delivering a double-chain break to the plasmid, lambda recombined as if the cells had become recD mutants. The ability of chi to alter lambda recombination in trans was reversed by overproducing the RecD subunit. These results indicate that chi can influence a recombination act without directly participating in it.}, }
@article {pmid7541534, year = {1995}, author = {Köppen, A and Krobitsch, S and Thoms, B and Wackernagel, W}, title = {Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {92}, number = {14}, pages = {6249-6253}, pmid = {7541534}, issn = {0027-8424}, mesh = {Bleomycin/pharmacology ; Crosses, Genetic ; DNA Nucleotidyltransferases/*biosynthesis ; Escherichia coli/*enzymology/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*biosynthesis/drug effects/*genetics ; *Integrases ; Kinetics ; Macromolecular Substances ; Mutagenesis ; Plasmids ; Recombinases ; *Recombination, Genetic ; Species Specificity ; }, abstract = {The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.}, }
@article {pmid7565099, year = {1995}, author = {Kuzminov, A}, title = {Collapse and repair of replication forks in Escherichia coli.}, journal = {Molecular microbiology}, volume = {16}, number = {3}, pages = {373-384}, doi = {10.1111/j.1365-2958.1995.tb02403.x}, pmid = {7565099}, issn = {0950-382X}, mesh = {Chromosomes, Bacterial/physiology ; DNA/genetics ; *DNA Damage ; DNA Polymerase I/physiology ; DNA Repair/*physiology ; DNA Replication/*physiology ; DNA, Bacterial/*biosynthesis ; DNA, Single-Stranded/genetics ; Escherichia coli/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/physiology ; *Models, Genetic ; Mutagenesis ; Rec A Recombinases/metabolism ; }, abstract = {Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.}, }
@article {pmid7892255, year = {1995}, author = {Biswas, I and Maguin, E and Ehrlich, SD and Gruss, A}, title = {A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {92}, number = {6}, pages = {2244-2248}, pmid = {7892255}, issn = {0027-8424}, mesh = {Base Composition ; Base Sequence ; *DNA Replication ; DNA, Bacterial/*genetics/metabolism ; Escherichia coli/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; Lactococcus lactis/*genetics ; Molecular Sequence Data ; Plasmids ; Species Specificity ; Substrate Specificity ; }, abstract = {Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.}, }
@article {pmid7836313, year = {1995}, author = {Horiuchi, T and Fujimura, Y}, title = {Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate.}, journal = {Journal of bacteriology}, volume = {177}, number = {3}, pages = {783-791}, doi = {10.1128/jb.177.3.783-791.1995}, pmid = {7836313}, issn = {0021-9193}, mesh = {*Chromosomes, Bacterial ; *DNA Replication ; Escherichia coli/*genetics/growth &amp; development ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/analysis ; Rec A Recombinases/analysis ; *Recombination, Genetic ; SOS Response, Genetics ; }, abstract = {To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural. While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E. coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction. The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model. We propose that the following sequential events may occur in both strains. A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally. this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication.}, }
@article {pmid7746848, year = {1995}, author = {Smith, GR and Amundsen, SK and Dabert, P and Taylor, AF}, title = {The initiation and control of homologous recombination in Escherichia coli.}, journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences}, volume = {347}, number = {1319}, pages = {13-20}, doi = {10.1098/rstb.1995.0003}, pmid = {7746848}, issn = {0962-8436}, support = {GM31693/GM/NIGMS NIH HHS/United States ; GM32194/GM/NIGMS NIH HHS/United States ; }, mesh = {Escherichia coli/*genetics ; *Recombination, Genetic ; }, abstract = {The chromosome of Escherichia coli recombines at low frequency when it is an intact circle but recombines at high frequency when it is broken, for example by X-rays, or when a linear DNA fragment is introduced into the cell during conjugation or transduction. The high recombinogenicity of double-strand (ds) DNA ends is attributable to RecBCD enzyme, which acts on ds DNA ends and is essential for recombination and ds DNA break repair. RecBCD enzyme initiates DNA unwinding at ds DNA ends, and its nuclease activity is controlled by Chi sites (5' G-C-T-G-G-T-G-G 3') in such a way that the enzyme produces a potent single-stranded DNA substrate for homologous pairing by RecA and single-stranded DNA binding proteins. We discuss a unifying model for recombination and ds DNA break repair, based upon the enzymic activities of these and other proteins and upon the behaviour of E. coli mutants altered in these proteins.}, }
@article {pmid8077199, year = {1994}, author = {Murphy, KC}, title = {Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme.}, journal = {The Journal of biological chemistry}, volume = {269}, number = {36}, pages = {22507-22516}, pmid = {8077199}, issn = {0021-9258}, support = {AI18234/AI/NIAID NIH HHS/United States ; }, mesh = {Bacteriophage P22/*enzymology ; Bacteriophage lambda/metabolism ; Chromatography ; Chromatography, DEAE-Cellulose ; Chromatography, Gel ; DNA Helicases/*metabolism ; DNA-Binding Proteins/metabolism ; Durapatite ; Escherichia coli/*enzymology ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/biosynthesis/isolation &amp; purification/*metabolism ; Kinetics ; Macromolecular Substances ; Mutagenesis, Site-Directed ; Plasmids ; Point Mutation ; Substrate Specificity ; Viral Proteins/metabolism/pharmacology ; }, abstract = {The biochemical properties of phage P22 Abc-modified RecBCD enzyme from Escherichia coli have been examined. RecBCD purified from a cell that expresses Abc (anti-RecBCD) contains all three RecBCD subunits and the 11.6-kDa Abc protein in equal stoichiometric amounts. Abc depresses the rate of RecBCD double-stranded DNA exonuclease, helicase, and ATPase activities about 3-4-fold, yet it has no effect on the rate of the single-stranded DNA exonuclease activity. Abc induces a slight increase in the ATP-independent single-stranded DNA endonuclease activity and does not induce dimerization of the RecBCD trimer. Abc-modified RecBCD helicase activity possesses reduced but significant processivity (10 kilobase pairs) relative to the native enzyme (30 kilobase pairs). In the absence of ATP, Abc-modified RecBCD shows a 2-4-fold higher affinity for double-stranded DNA ends. The RecBCD-binding Gam protein from bacteriophage lambda inhibits binding of both native and Abc-modified RecBCD to double-stranded DNA ends. Finally, unlike the native enzyme, the nonspecific nuclease activity of Abc-modified RecBCD is not suppressed by Chi sites in vitro. These findings are discussed in terms of the recombination-deficient phenotype of cells expressing Abc in vivo and the relationship between Abc-modified RecBCD and two mutant RecBCD's previously characterized: the RecBCD-K117Q and RecB2109CD mutant enzymes.}, }
@article {pmid7968921, year = {1994}, author = {Kowalczykowski, SC and Dixon, DA and Eggleston, AK and Lauder, SD and Rehrauer, WM}, title = {Biochemistry of homologous recombination in Escherichia coli.}, journal = {Microbiological reviews}, volume = {58}, number = {3}, pages = {401-465}, pmid = {7968921}, issn = {0146-0749}, support = {AI-18987/AI/NIAID NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Escherichia coli/*genetics/metabolism ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; *Models, Genetic ; Rec A Recombinases/genetics/*metabolism ; Recombination, Genetic/*genetics ; }, abstract = {Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.}, }
@article {pmid8051022, year = {1994}, author = {Zaman, MM and Boles, TC}, title = {Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli.}, journal = {Journal of bacteriology}, volume = {176}, number = {16}, pages = {5093-5100}, pmid = {8051022}, issn = {0021-9193}, mesh = {Bacterial Proteins/genetics ; Base Sequence ; DNA, Bacterial/*genetics ; DNA-Binding Proteins/genetics ; Escherichia coli/*enzymology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/deficiency/*metabolism ; Molecular Sequence Data ; Molecular Weight ; Oligodeoxyribonucleotides/chemistry ; *Plasmids ; Rec A Recombinases/genetics ; Recombination, Genetic ; }, abstract = {Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.}, }
@article {pmid8045897, year = {1994}, author = {Horiuchi, T and Fujimura, Y and Nishitani, H and Kobayashi, T and Hidaka, M}, title = {The DNA replication fork blocked at the Ter site may be an entrance for the RecBCD enzyme into duplex DNA.}, journal = {Journal of bacteriology}, volume = {176}, number = {15}, pages = {4656-4663}, pmid = {8045897}, issn = {0021-9193}, mesh = {Bacterial Proteins/metabolism ; Base Sequence ; Chromosomes, Bacterial ; DNA Helicases/*metabolism ; *DNA Replication ; DNA, Bacterial/*biosynthesis ; DNA, Circular ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Models, Genetic ; Molecular Sequence Data ; Nucleic Acid Conformation ; Recombination, Genetic ; tau Proteins/metabolism ; }, abstract = {In Escherichia coli, eight kinds of chromosome-derived DNA fragments (named Hot DNA) were found to exhibit homologous recombinational hotspot activity, with the following properties. (i) The Hot activities of all Hot DNAs were enhanced extensively under RNase H-defective (rnh) conditions. (ii) Seven Hot DNAs were clustered at the DNA replication terminus region on the E. coli chromosome and had Chi activities (H. Nishitani, M. Hidaka, and T. Horiuchi, Mol. Gen. Genet. 240:307-314, 1993). Hot activities of HotA, -B, and -C, the locations of which were close to three DNA replication terminus sites, the TerB, -A, and -C sites, respectively, disappeared when terminus-binding (Tau or Tus) protein was defective, thereby suggesting that their Hot activities are termination event dependent. Other Hot groups showed termination-independent Hot activities. In addition, at least HotA activity proved to be dependent on a Chi sequence, because mutational destruction of the Chi sequence on the HotA DNA fragment resulted in disappearance of the HotA activity. The HotA activity which had disappeared was reactivated by insertion of a new, properly oriented Chi sequence at the position between the HotA DNA and the TerB site. On the basis of these observations and positional and orientational relationships between the Chi and the Ter sequences, we propose a model in which the DNA replication fork blocked at the Ter site provides an entrance for the RecBCD enzyme into duplex DNA.}, }
@article {pmid8021190, year = {1994}, author = {Miesel, L and Roth, JR}, title = {Salmonella recD mutations increase recombination in a short sequence transduction assay.}, journal = {Journal of bacteriology}, volume = {176}, number = {13}, pages = {4092-4103}, pmid = {8021190}, issn = {0021-9193}, support = {GM27068/GM/NIGMS NIH HHS/United States ; T32-GM07464-15/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage P22/genetics ; DNA Repair ; Escherichia coli/genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics ; Genetic Complementation Test ; Models, Genetic ; Mutagenesis, Insertional ; *Mutation ; Plasmids/genetics ; *Recombination, Genetic ; Salmonella typhimurium/*genetics ; *Transduction, Genetic ; }, abstract = {We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease.}, }
@article {pmid8026461, year = {1994}, author = {Kuzminov, A and Schabtach, E and Stahl, FW}, title = {Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.}, journal = {The EMBO journal}, volume = {13}, number = {12}, pages = {2764-2776}, pmid = {8026461}, issn = {0261-4189}, mesh = {Base Sequence ; Cosmids/metabolism ; *DNA Damage ; DNA, Bacterial/*metabolism/ultrastructure ; DNA-Binding Proteins/metabolism ; Endodeoxyribonucleases/metabolism ; Escherichia coli/*metabolism ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Models, Genetic ; Molecular Sequence Data ; Nucleic Acid Conformation ; Protein Binding ; Rec A Recombinases/*metabolism ; Recombination, Genetic ; }, abstract = {In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.}, }
@article {pmid8159691, year = {1994}, author = {Dixon, DA and Churchill, JJ and Kowalczykowski, SC}, title = {Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {91}, number = {8}, pages = {2980-2984}, pmid = {8159691}, issn = {0027-8424}, support = {GM41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Helicases/metabolism ; DNA, Bacterial/genetics ; Deoxyribonucleases/metabolism ; Escherichia coli/enzymology/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Exonucleases/metabolism ; Genes, Regulator ; In Vitro Techniques ; Mutation ; *Recombination, Genetic ; *Regulatory Sequences, Nucleic Acid ; }, abstract = {Genetic recombination in Escherichia coli is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (chi) sites (5'-GCTGGTGG-3'). Previously, it was shown that chi acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with chi sites can also result in an inactivation of helicase activity of RecBCD. The unwinding of double-stranded DNA-containing chi sites, under conditions of limiting Mg2+ ion, results in the reversible inactivation of RecBCD; addition of excess Mg2+ to the reaction reactivates all activities of RecBCD. Inactivation is the consequence of a chi-dependent modification of RecBCD that appears to result from an inability of the chi-modified RecBCD to reinitiate unwinding of intact DNA molecules. This characteristic behavior of RecBCD and chi is displayed by the reconstituted RecBC (i.e., without the RecD subunit), except that it is not dependent on chi interaction. This biochemical similarity between the chi-modified RecBCD and RecBC enzymes implies that recognition of chi results in a dissociation or functional inactivation of RecD subunit and lends support to the hypothesis that interaction with chi results in ejection of the RecD subunit.}, }
@article {pmid8143797, year = {1994}, author = {Smith, GR}, title = {Hotspots of homologous recombination.}, journal = {Experientia}, volume = {50}, number = {3}, pages = {234-241}, pmid = {8143797}, issn = {0014-4754}, support = {GM31693/GM/NIGMS NIH HHS/United States ; }, mesh = {Binding Sites ; Escherichia coli/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; Schizosaccharomyces/*genetics ; }, abstract = {Homologous recombination occurs at higher than average frequency at and near hotspots. Hotspots are special nucleotide sequences recognized by proteins that promote, directly or indirectly, a rate limiting step of recombination. This review focuses on two well-studied examples, the Chi sites of the bacterium Escherichia coli and the M26 site of the fission yeast Schizosaccharomyces pombe. Chi, 5' G-C-T-G-G-T-G-G 3', is recognized by the RecBCD enzyme, which nicks the DNA near Chi and produces a 3'-ended single-stranded DNA 'tail'; this tail is a potent substrate for homologous pairing by RecA and single-stranded DNA binding proteins. M26, 5' A-T-G-A-C-G-T 3', is recognized by a heterodimeric protein and stimulates, by an as-yet-unknown mechanism, meiotic recombination at and near the ade6 gene. Additional hotspots in bacteria, fungi, and mammals enhance recombination directly or indirectly via a variety of mechanisms. Although hotspots are widespread among organisms, the biological role of their localized enhancement of recombination remains a matter of speculation.}, }
@article {pmid8143794, year = {1994}, author = {Kowalczykowski, SC}, title = {In vitro reconstitution of homologous recombination reactions.}, journal = {Experientia}, volume = {50}, number = {3}, pages = {204-215}, pmid = {8143794}, issn = {0014-4754}, support = {AI-18987/AI/NIAID NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA/metabolism ; DNA Helicases/*metabolism ; DNA-Binding Proteins/metabolism ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Rec A Recombinases/metabolism ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; }, abstract = {The proteins essential to homologous recombination in E. coli have been purified and their individual activities have been identified, permitting biochemical reconstitution of steps that comprise the cellular recombination process. This review focuses on the biochemical events responsible for the initiation and homologous pairing steps of genetic recombination. The properties of an in vitro recombination reaction that requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(chi), are described. The recBCD enzyme serves as the initiator of this reaction; its DNA helicase activity produces single-stranded DNA that is used by the recA protein to promote homologous pairing and DNA strand invasion of supercoiled (recipient) DNA. The SSB protein acts to trap the single-stranded DNA produced by recBCD enzyme and to facilitate pairing by the recA protein. The chi regulatory sequence acts in cis by attenuating the nuclease, but not the helicase, activity of recBCD enzyme. This attenuation assures the preservation of ssDNA produced by the DNA helicase activity and is responsible for the simulation in vitro and, presumably, in vivo. The attenuation of nuclease activity by chi results in the loss or functional inactivation of the recD subunit.}, }
@article {pmid7893137, year = {1994}, author = {Myers, RS and Stahl, FW}, title = {Chi and the RecBC D enzyme of Escherichia coli.}, journal = {Annual review of genetics}, volume = {28}, number = {}, pages = {49-70}, doi = {10.1146/annurev.ge.28.120194.000405}, pmid = {7893137}, issn = {0066-4197}, support = {5F32GM14196/GM/NIGMS NIH HHS/United States ; GM33677/GM/NIGMS NIH HHS/United States ; }, mesh = {Escherichia coli/enzymology/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/metabolism ; Mutation ; Oligodeoxyribonucleotides ; *Recombination, Genetic ; Substrate Specificity ; }, abstract = {The products of genes recB and recC are responsible for conjugal recombination and for the repair of chromosomal double chain breaks in Escherichia coli. The product of the recD gene, which combines with the RecB and RecC proteins to comprise the RecBCD enzyme, is not required for either recombination or repair. On the contrary, RecBCD enzyme is a potent exonuclease which inhibits recombination by destroying linear DNA. The RecD Ejection model supposes that RecBCD enzyme enters DNA at a double-chain end and travels destructively along the DNA until (typically) it encounters the recombination hotspot sequence chi. Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer. With the loss of the RecD subunit from the enzyme, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase. Thus, the RecD Ejection model proposes that chi participates in recombination by acting as a toggle to convert RecBCD (a powerful exonuclease) to RecBC(D-) (a recombinase). We review the properties of RecBCD and its cognate site chi, including recent results that support both the RecD Ejection model and the view that chi plays only an indirect role in recombination.}, }
@article {pmid8346244, year = {1993}, author = {Tummuru, MK and Blaser, MJ}, title = {Rearrangement of sapA homologs with conserved and variable regions in Campylobacter fetus.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {90}, number = {15}, pages = {7265-7269}, pmid = {8346244}, issn = {0027-8424}, support = {R01A1-24145//PHS HHS/United States ; }, mesh = {Antigens, Bacterial/*genetics ; Bacterial Proteins/*genetics ; Base Sequence ; Campylobacter fetus/*genetics ; Cloning, Molecular ; DNA, Bacterial/genetics ; Gene Rearrangement ; *Genes, Bacterial ; *Membrane Glycoproteins ; Molecular Sequence Data ; Phenotype ; Recombination, Genetic ; Restriction Mapping ; Sequence Alignment ; Sequence Homology, Nucleic Acid ; Species Specificity ; }, abstract = {The Campylobacter fetus surface-layer (S-layer) proteins mediate both complement resistance and antigenic variation in mammalian hosts. Wild-type strain 23D possesses the sapA gene, which encodes a 97-kDa S-layer protein, and several sapA homologs are present in both wild-type and mutant strains. Here we report that a cloned silent gene (sapA1) in C. fetus can express a functional full-length S-layer protein in Escherichia coli. Analysis of sapA and sapA1 and partial analysis of sapA2 indicate that a block of approximately 600 bp beginning upstream and continuing into the open reading frames is completely conserved, and then the sequences diverge completely, but immediately downstream of each gene is another conserved 50-bp sequence. Conservation of sapA1 among strains, the presence of a putative Chi (RecBCD recognition) site upstream of sapA, sapA1, and sapA2, and the sequence identities of the sapA genes suggest a system for homologous recombination. Comparison of the wild-type strain (23D) with a phenotypic variant (23D-11) indicates that variation is associated with removal of the divergent region of sapA from the expression locus and exchange with a corresponding region from a sapA homolog. We propose that site-specific reciprocal recombination between sapA homologs leads to expression of divergent S-layer proteins as one of the mechanisms that C. fetus uses for antigenic variation.}, }
@article {pmid8390578, year = {1993}, author = {Eggleston, AK and Kowalczykowski, SC}, title = {The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro.}, journal = {Journal of molecular biology}, volume = {231}, number = {3}, pages = {621-633}, doi = {10.1006/jmbi.1993.1314}, pmid = {8390578}, issn = {0022-2836}, support = {AI-18987/AI/NIAID NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; NRSA GM-08061/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/metabolism ; DNA/metabolism ; DNA Helicases/drug effects/*genetics/metabolism ; Escherichia coli/enzymology ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/drug effects/*genetics/metabolism ; Kinetics ; Magnesium/pharmacology ; *Mutation ; Sodium Chloride/pharmacology ; }, abstract = {The Escherichia coli recB2109CD enzyme displays a defect in homologous recombination. In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in chi-dependent nicking activity. To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB2109CD enzyme to unwind dsDNA was examined. The mutant enzyme is able to unwind DNA but has a kcat which is one-third that of the wild-type enzyme. While the Km for DNA ends of the wild-type and mutant enzymes at low NaCl concentrations are essentially equivalent, the Km for ATP of recB2109CD enzyme is nearly six times greater. The processivity of unwinding (i.e. the average length of DNA unwound before recB2109CD enzyme dissociates from the DNA substrate) at 1 mM-Mg2+ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end. In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB2109CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme. We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the chi-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.}, }
@article {pmid8390577, year = {1993}, author = {Eggleston, AK and Kowalczykowski, SC}, title = {Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity.}, journal = {Journal of molecular biology}, volume = {231}, number = {3}, pages = {605-620}, doi = {10.1006/jmbi.1993.1313}, pmid = {8390577}, issn = {0022-2836}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; NRSA GM-08061/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/metabolism ; DNA Helicases/*genetics/metabolism ; DNA, Single-Stranded/metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/enzymology ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/metabolism ; Kinetics ; Magnesium/metabolism ; *Mutation ; Substrate Specificity ; }, abstract = {RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced approximately 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at chi sites, which are hotspots for genetic recombination. Since this interaction with chi significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.}, }
@article {pmid8384931, year = {1993}, author = {Dixon, DA and Kowalczykowski, SC}, title = {The recombination hotspot chi is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme.}, journal = {Cell}, volume = {73}, number = {1}, pages = {87-96}, doi = {10.1016/0092-8674(93)90162-j}, pmid = {8384931}, issn = {0092-8674}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {DNA Helicases/*metabolism ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Models, Genetic ; Recombination, Genetic/*genetics ; Regulatory Sequences, Nucleic Acid ; }, abstract = {The RecBCD enzyme is a multifunctional enzyme that is essential for homologous recombination in E. coli. In vitro, the RecBCD enzyme degrades linear double-stranded DNA nonspecifically during the process of unwinding the double-stranded DNA. Here we demonstrate that this DNA degradation is asymmetric, with the strand that is 3' terminal at the entry site of RecBCD enzyme being degraded much more vigorously than the 5' terminal strand. Furthermore, interaction with the recombination hotspot chi causes an attenuation of the nuclease activity but not of the helicase activity and is accompanied by a pause of RecBCD enzyme at the chi site. These results demonstrate that chi is a unique regulatory element that acts by controlling the degradative function of RecBCD enzyme and, thereby, enhancing its recombination function.}, }
@article {pmid8387145, year = {1993}, author = {Kooistra, J and Haijema, BJ and Venema, G}, title = {The Bacillus subtilis addAB genes are fully functional in Escherichia coli.}, journal = {Molecular microbiology}, volume = {7}, number = {6}, pages = {915-923}, doi = {10.1111/j.1365-2958.1993.tb01182.x}, pmid = {8387145}, issn = {0950-382X}, mesh = {Bacillus subtilis/*genetics/metabolism ; Bacterial Proteins/genetics/*metabolism ; Bacteriophages/genetics ; DNA Helicases/genetics/metabolism ; DNA Repair ; DNA, Viral/genetics ; Escherichia coli/*metabolism ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/metabolism ; Genetic Complementation Test ; Genetic Vectors ; Plasmids ; Recombinant Fusion Proteins/genetics/*metabolism ; Recombination, Genetic ; Species Specificity ; Transformation, Bacterial ; }, abstract = {An Escherichia coli recBCD deletion mutant was transformed with plasmids containing the Bacillus subtilis add genes. The transformants had relatively high ATP-dependent exonuclease- and ATP-dependent helicase activities, and their viability, the ability to repair u.v.-damaged DNA and the recombination in conjugation were nearly completely restored. The B. subtilis Add enzyme did not show Chi-activity in phage lambda recombination. The individual B. subtilis Add proteins were not able to form an enzymatically active complex with the E. coli RecB,C,D proteins, and they could not complement the recB,C,D deficiency. Evidence is presented that only two subunits are involved in the B. subtilis ATP-dependent exonuclease. This is in contrast to E. coli in which the RecBCD enzyme consists of three subunits.}, }
@article {pmid8383665, year = {1993}, author = {Murphy, KC and Lewis, LJ}, title = {Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity.}, journal = {Journal of bacteriology}, volume = {175}, number = {6}, pages = {1756-1766}, pmid = {8383665}, issn = {0021-9193}, support = {AI18234/AI/NIAID NIH HHS/United States ; }, mesh = {Bacteriophage P22/*enzymology/genetics ; Base Sequence ; Binding Sites ; Conjugation, Genetic ; DNA Helicases/genetics/*metabolism ; DNA, Bacterial/metabolism ; Escherichia coli/*genetics/growth &amp; development/metabolism/radiation effects ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/*metabolism ; Molecular Sequence Data ; Nalidixic Acid/pharmacology ; Plasmids ; Recombination, Genetic ; SOS Response, Genetics ; Ultraviolet Rays ; }, abstract = {Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes. Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants. Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold. Abc2 induces E. coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests. Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does). Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants. These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.}, }
@article {pmid1465442, year = {1992}, author = {Dabert, P and Ehrlich, SD and Gruss, A}, title = {Chi sequence protects against RecBCD degradation of DNA in vivo.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {89}, number = {24}, pages = {12073-12077}, pmid = {1465442}, issn = {0027-8424}, mesh = {Base Sequence ; DNA Replication ; DNA, Bacterial/*genetics ; Escherichia coli/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Molecular Sequence Data ; Oligodeoxyribonucleotides/chemistry ; Plasmids ; Rec A Recombinases/*metabolism ; Recombination, Genetic ; }, abstract = {RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.}, }
@article {pmid1459441, year = {1992}, author = {Holbeck, SL and Smith, GR}, title = {Chi enhances heteroduplex DNA levels during recombination.}, journal = {Genetics}, volume = {132}, number = {4}, pages = {879-891}, pmid = {1459441}, issn = {0016-6731}, support = {GM31693/GM/NIGMS NIH HHS/United States ; GM32194/GM/NIGMS NIH HHS/United States ; T32 ES07032/ES/NIEHS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/genetics ; Base Sequence ; DNA, Bacterial/genetics ; DNA, Viral/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics ; Rec A Recombinases/genetics ; *Recombination, Genetic ; }, abstract = {The major pathway of homologous recombination in Escherichia coli, the RecBCD pathway, is stimulated by Chi sites. To determine whether Chi enhances an early or late step in recombination, we measured formation of heteroduplex DNA (hDNA) in extracts of lambda-infected E. coli. Chi elevated hDNA levels in these extracts, supporting a role for Chi early (before hDNA formation) in recombination. RecA protein and RecBCD enzyme were both necessary for detection of hDNA, indicating that they, too, act early. Analysis of a panel of recBCD mutants indicated that Chi-nicking activity was needed for Chi's stimulation of hDNA formation. These results support a previously proposed model of recombination. Further results suggested that RecBCD enzyme has an additional role late in recombination.}, }
@article {pmid1322885, year = {1992}, author = {Rinken, R and Thomas, B and Wackernagel, W}, title = {Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.}, journal = {Journal of bacteriology}, volume = {174}, number = {16}, pages = {5424-5429}, pmid = {1322885}, issn = {0021-9193}, mesh = {DNA Helicases/*metabolism ; DNA, Bacterial/*metabolism ; DNA, Viral/metabolism ; Escherichia coli/*metabolism ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/*metabolism ; Mutation ; Phenotype ; T-Phages/metabolism ; Thymidine/metabolism ; }, abstract = {Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.}, }
@article {pmid1618858, year = {1992}, author = {Masterson, C and Boehmer, PE and McDonald, F and Chaudhuri, S and Hickson, ID and Emmerson, PT}, title = {Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits.}, journal = {The Journal of biological chemistry}, volume = {267}, number = {19}, pages = {13564-13572}, pmid = {1618858}, issn = {0021-9258}, mesh = {Adenosine Triphosphate/metabolism ; Chromatography, Liquid ; DNA, Bacterial/metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/*enzymology ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics/isolation &amp; purification/*metabolism ; Mutagenesis, Site-Directed ; }, abstract = {The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.}, }
@article {pmid1535156, year = {1992}, author = {Taylor, AF and Smith, GR}, title = {RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {89}, number = {12}, pages = {5226-5230}, pmid = {1535156}, issn = {0027-8424}, mesh = {Bacteriophage lambda/genetics ; Conjugation, Genetic ; DNA, Bacterial/genetics/isolation &amp; purification/*metabolism ; DNA, Viral/genetics/isolation &amp; purification/*metabolism ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Kinetics ; Plasmids ; *Recombination, Genetic ; Substrate Specificity ; }, abstract = {During its unidirectional unwinding of DNA, RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia coli. We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites. Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected. These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.}, }
@article {pmid1310990, year = {1992}, author = {Roman, LJ and Eggleston, AK and Kowalczykowski, SC}, title = {Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme.}, journal = {The Journal of biological chemistry}, volume = {267}, number = {6}, pages = {4207-4214}, pmid = {1310990}, issn = {0021-9258}, support = {AI-18987/AI/NIAID NIH HHS/United States ; GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Diphosphate/metabolism ; Adenosine Triphosphate/metabolism ; Cations, Divalent ; Cations, Monovalent ; DNA Helicases/*metabolism ; DNA, Bacterial/*metabolism ; Electrophoresis, Gel, Pulsed-Field ; Escherichia coli/*enzymology ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Fluorescence Polarization ; }, abstract = {A fluorescence assay was used to measure the processivity of Escherichia coli recBCD enzyme helicase activity. Under standard conditions, recBCD enzyme unwinds an average of 30 +/- 3.2 kilobase pairs (kb)/DNA end before dissociating. The average processivity (P obs) of DNA unwinding under these conditions is 0.99997, indicating that the probability of unwinding another base pair is 30,000-fold greater than the probability of dissociating from the double-stranded DNA. The average number of base pairs unwound per binding event (N) is sensitive to both mono- and divalent salt concentration and ranges from 36 kb at 80 mM NaCl to 15 kb at 280 mM NaCl. The processivity of unwinding increases in a hyperbolic manner with increasing ATP concentration, yielding a KN value for ATP of 41 +/- 9 microM and a limiting value of 32 +/- 1.8 kb/end for the number of base pairs unwound. The importance of the processivity of recBCD enzyme helicase activity to the recBCD enzyme-dependent stimulation of recombination at Chi sites observed in vivo is discussed.}, }
@article {pmid1310498, year = {1992}, author = {Rinken, R and Wackernagel, W}, title = {Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.}, journal = {Journal of bacteriology}, volume = {174}, number = {4}, pages = {1172-1178}, pmid = {1310498}, issn = {0021-9193}, mesh = {DNA Helicases/*metabolism ; Escherichia coli/drug effects/*genetics/radiation effects ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; Mitomycins/*toxicity ; Mutation/genetics ; Nalidixic Acid/*toxicity ; Plasmids/genetics ; Recombination, Genetic/*drug effects/genetics/radiation effects ; SOS Response, Genetics/*genetics ; Sequence Homology, Nucleic Acid ; }, abstract = {Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.}, }
@article {pmid1836442, year = {1991}, author = {Hagemann, AT and Rosenberg, SM}, title = {Chain bias in Chi-stimulated heteroduplex patches in the lambda ren gene is determined by the orientation of lambda cos.}, journal = {Genetics}, volume = {129}, number = {3}, pages = {611-621}, pmid = {1836442}, issn = {0016-6731}, support = {GM07413-09/GM/NIGMS NIH HHS/United States ; GM3367/GM/NIGMS NIH HHS/United States ; GM41747-01/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*genetics ; DNA, Viral/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*physiology ; Genes, Regulator ; Genes, Viral ; *Recombination, Genetic ; Regulatory Sequences, Nucleic Acid ; Viral Structural Proteins/genetics ; }, abstract = {Heteroduplex patch recombinants have received information in one DNA chain but have not recombined flanking markers. Evidence regarding which chain is exchanged bears on the structure of recombination intermediates. The direction of travel along DNA of RecBCD recombinase, the central enzyme in the Escherichia coli RecBCD pathway of homologous recombination, is determined in phage lambda by the orientation of the packaging origin, cos. cos is a double-chain cut site which serves as a preferred entry site for RecBCD. Using partially denaturing gels to resolve heteroduplex molecules, we have examined patch recombinants at the lambda ren gene. We report that the transferred information in Chi-stimulated patches at ren can occur on either chain, but is biased to the chain ending 5' at the right of the lambda map (the lambda r chain) in phage carrying cos in its normal orientation. The chain bias switches in favor of the chain that ends 3' at the right (the lambda l chain) when RecBCD travel direction is reversed by inverting cos. We entertain models that accommodate these and other results pertaining to the structure of RecBCD-mediated recombinants.}, }
@article {pmid1653221, year = {1991}, author = {Murphy, KC}, title = {Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme.}, journal = {Journal of bacteriology}, volume = {173}, number = {18}, pages = {5808-5821}, pmid = {1653221}, issn = {0021-9193}, support = {AI18234/AI/NIAID NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; Bacteriophage lambda/*genetics ; Base Sequence ; Cloning, Molecular ; Conjugation, Genetic ; DNA Helicases/*antagonists &amp; inhibitors ; DNA Repair ; DNA Replication ; DNA, Bacterial/radiation effects ; DNA, Viral/genetics ; DNA-Binding Proteins/*pharmacology ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*antagonists &amp; inhibitors/metabolism ; Molecular Sequence Data ; Recombination, Genetic/*drug effects ; Ultraviolet Rays ; Viral Proteins/*pharmacology ; }, abstract = {The lambda Gam protein was isolated from cells containing a Gam-producing plasmid. The purified Gam protein was found to bind to RecBCD without displacing any of its subunits. Gam was shown to inhibit all known enzymatic activities of RecBCD: ATP-dependent single- and double-stranded DNA exonucleases, ATP-independent single-stranded endonuclease, and the ATP-dependent helicase. When produced in vivo, Gam inhibited chi-activated recombination in lambda red gam crosses but had little effect on the host's ability to act as a recipient in conjugational recombination. These experiments suggest that RecBCD possesses an additional "unknown" activity that is resistant to or induced by Gam. Additionally, the expression of Gam in recD mutants sensitizes the host to UV irradiation, indicating that Gam alters one or more of the in vivo activities of RecBC(D-).}, }
@article {pmid1855256, year = {1991}, author = {Dixon, DA and Kowalczykowski, SC}, title = {Homologous pairing in vitro stimulated by the recombination hotspot, Chi.}, journal = {Cell}, volume = {66}, number = {2}, pages = {361-371}, doi = {10.1016/0092-8674(91)90625-9}, pmid = {1855256}, issn = {0092-8674}, support = {GM-41347/GM/NIGMS NIH HHS/United States ; }, mesh = {Base Sequence ; DNA, Bacterial/*genetics/metabolism ; DNA-Binding Proteins/metabolism ; Escherichia coli/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; Kinetics ; Molecular Sequence Data ; Oligonucleotide Probes ; Plasmids ; Rec A Recombinases/metabolism ; *Recombination, Genetic ; Restriction Mapping ; Substrate Specificity ; }, abstract = {Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5'-GCT-GGTGG-3'. We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot. Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA. The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi. This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3' end for efficient joint molecule formation. Action at Chi generates invasive ssDNA from the 5' but not the 3' side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site. These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.}, }
@article {pmid1655050, year = {1991}, author = {Rinken, R and de Vries, J and Weichenhan, D and Wackernagel, W}, title = {The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.}, journal = {Biochimie}, volume = {73}, number = {4}, pages = {375-384}, doi = {10.1016/0300-9084(91)90104-9}, pmid = {1655050}, issn = {0300-9084}, mesh = {Chromosome Mapping ; Chromosomes, Bacterial ; DNA Helicases/*genetics/physiology ; Escherichia coli/enzymology/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/physiology ; Genes, Bacterial ; Mutation ; Proteus mirabilis/enzymology/*genetics ; Rec A Recombinases/*genetics ; *Recombination, Genetic ; Serratia marcescens/enzymology/*genetics ; }, abstract = {The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.}, }
@article {pmid2087220, year = {1990}, author = {Meyer, RR and Laine, PS}, title = {The single-stranded DNA-binding protein of Escherichia coli.}, journal = {Microbiological reviews}, volume = {54}, number = {4}, pages = {342-380}, pmid = {2087220}, issn = {0146-0749}, support = {GM31196/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Sequence ; DNA, Bacterial/genetics/metabolism ; DNA-Binding Proteins/*chemistry/genetics/metabolism ; Escherichia coli/*genetics ; Molecular Sequence Data ; Protein Conformation ; Regulatory Sequences, Nucleic Acid ; }, abstract = {The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.}, }
@article {pmid2249753, year = {1990}, author = {Stahl, FW and Thomason, LC and Siddiqi, I and Stahl, MM}, title = {Further tests of a recombination model in which chi removes the RecD subunit from the RecBCD enzyme of Escherichia coli.}, journal = {Genetics}, volume = {126}, number = {3}, pages = {519-533}, pmid = {2249753}, issn = {0016-6731}, support = {GM 33677/GM/NIGMS NIH HHS/United States ; }, mesh = {Crosses, Genetic ; Escherichia coli/enzymology/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/metabolism ; *Models, Genetic ; Plasmids ; *Recombination, Genetic ; }, abstract = {When one of two infecting lambda phage types in a replication-blocked cross is chi + and DNA packaging is divorced from the RecBCD-chi interaction, complementary chi-stimulated recombinants are recovered equally in mass lysates only if the chi + parent is in excess in the infecting parental mixture. Otherwise, the chi 0 recombinant is recovered in excess. This observation implies that, along with the chi 0 chromosome, two chi + parent chromosomes are involved in the formation of each chi + recombinant. The trimolecular nature of chi +-stimulated recombination is manifest in recombination between lambda and a plasmid. When lambda recombines with a plasmid via the RecBCD pathway, the resulting chromosome has an enhanced probability of undergoing lambda x lambda recombination in the interval into which the plasmid was incorporated. These two observations support a model in which DNA is degraded by Exo V from cos, the sequence that determines the end of packaged lambda DNA and acts as point of entry for RecBCD enzyme, to chi, the DNA sequence that stimulates the RecBCD enzyme to effect recombination. The model supposes that chi acts by ejecting the RecD subunit from the RecBCD enzyme with two consequences. (1) ExoV activity is blocked leaving a highly recombinagenic, frayed duplex end near chi, and (2) as the enzyme stripped of the RecD subunit travels beyond chi it is competent to catalyze reciprocal recombination.}, }
@article {pmid2172076, year = {1990}, author = {Amundsen, SK and Neiman, AM and Thibodeaux, SM and Smith, GR}, title = {Genetic dissection of the biochemical activities of RecBCD enzyme.}, journal = {Genetics}, volume = {126}, number = {1}, pages = {25-40}, pmid = {2172076}, issn = {0016-6731}, support = {GM12077/GM/NIGMS NIH HHS/United States ; GM31693/GM/NIGMS NIH HHS/United States ; GM32194/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/metabolism ; Coliphages/growth &amp; development ; DNA Helicases/*genetics/metabolism ; DNA Transposable Elements ; Drug Resistance, Microbial/genetics ; Escherichia coli/enzymology/*genetics/growth &amp; development ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/metabolism ; Genetic Complementation Test ; Mitomycin ; Mitomycins/pharmacology ; Mutation ; Plasmids ; *Recombination, Genetic ; }, abstract = {RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.}, }
@article {pmid2559208, year = {1989}, author = {McKittrick, NH and Smith, GR}, title = {Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria.}, journal = {Journal of molecular biology}, volume = {210}, number = {3}, pages = {485-495}, doi = {10.1016/0022-2836(89)90125-3}, pmid = {2559208}, issn = {0022-2836}, support = {GM31693/GM/NIGMS NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/metabolism ; Bacteriophage lambda/genetics ; Cloning, Molecular ; DNA Helicases/genetics ; Enterobacteriaceae/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics/metabolism ; Genetic Complementation Test ; Plasmids ; *Recombination, Genetic ; Viral Plaque Assay ; Virus Replication ; }, abstract = {Chi sites, 5'G-C-T-G-G-T-G-G-3', enhance homologous recombination in Escherichia coli and are activated by the RecBCD enzyme. To test the ability of Chi to be activated by analogous enzymes from other bacteria, we cloned recBCD-like genes from diverse bacteria into an E. coli recBCD deletion mutant. Clones from seven species of enteric bacteria conferred to this deletion mutant recombination proficiency, Chi hotspot activity in lambda Red- Gam- vegetative crosses, and RecBCD enzyme activities, including Chi-dependent DNA strand cleavage. Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. These results imply that Chi has been conserved as a recombination-promoting signal for RecBCD-like enzymes in enteric bacteria but not in more distantly related bacteria such as Pseudomonas spp. We discuss the possibility that other, presently unknown, nucleotide sequences serve the same function as Chi in Pseudomonas spp.}, }
@article {pmid2560121, year = {1989}, author = {Weichenhan, D and Wackernagel, W}, title = {Functional analyses of Proteus mirabilis wild-type and mutant RecBCD enzymes in Escherichia coli reveal a new mutant phenotype.}, journal = {Molecular microbiology}, volume = {3}, number = {12}, pages = {1777-1784}, doi = {10.1111/j.1365-2958.1989.tb00163.x}, pmid = {2560121}, issn = {0950-382X}, mesh = {Chromosome Mapping ; *DNA Helicases ; DNA, Bacterial/genetics ; Escherichia coli/enzymology/*genetics/radiation effects ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics ; *Genes, Regulator ; Molecular Weight ; Mutation ; Phenotype ; Proteus mirabilis/*genetics ; Transcription, Genetic ; Ultraviolet Rays ; }, abstract = {Cloned into Escherichia coli the recB, recC and recD genes of Proteus mirabilis produce a recBCD enzyme (exoV) functional in recombination and DNA repair. The direction of transcription of recB, recC and recD, the sizes of the enzyme subunits, and their composition in the active enzyme are similar to that observed for the E. coli enzyme. In lambda crosses, the P. mirabilis enzyme has only about 40% of the Chi activity of the E. coli enzyme. The recBCD genes were also cloned from an exoV mutant of P. mirabilis which is u.v.-sensitive and partly deficient in exoV. The defect was attributed to the recB gene by complementation studies. In a recBCD deletion strain of E. coli, the enzyme from the mutant produced 40% of conjugational recombinants and had retained about 25% of Chi activity. However, it did not restore normal DNA repair, cell viability or recombination in lambda crosses and P1 transduction. The new mutant phenotype is discussed in the light of the assumption that prokaryotic recBCD enzymes can promote recombination in a Chi-dependent and a Chi-independent manner.}, }
@article {pmid2530132, year = {1989}, author = {Cheng, KC and Smith, GR}, title = {Distribution of Chi-stimulated recombinational exchanges and heteroduplex endpoints in phage lambda.}, journal = {Genetics}, volume = {123}, number = {1}, pages = {5-17}, pmid = {2530132}, issn = {0016-6731}, support = {GM31693/GM/NIGMS NIH HHS/United States ; T32 GM07187/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*genetics ; Crossing Over, Genetic ; Escherichia coli ; Exodeoxyribonucleases/physiology ; Genetic Markers ; *Recombination, Genetic ; }, abstract = {The recombination hotspot Chi, 5' G-C-T-G-G-T-G-G 3', stimulates the RecBCD recombination pathway of Escherichia coli. We have determined, with precision greater than previously reported, the distribution of Chi-stimulated exchanges around a Chi site in phage lambda. Crosses of lambda phages with single base-pair mutations surrounding a Chi site were conducted in and analyzed on mismatch correction-impaired hosts to preserve heteroduplex mismatches for analysis. Among phages recombinant for flanking markers, Chi stimulated exchanges most intensely in the intervals immediately adjacent to the Chi site, both to its right and to its left. Stimulation fell off abruptly to the right but gradually to the left (with respect to the orientation of the Chi sequence written above). We have also determined that Chi stimulated the formation of heteroduplex DNA, which frequently had one endpoint to the right of Chi and the other endpoint to the left. These data support a model of Chi-stimulated recombination in which RecBCD enzyme cuts DNA immediately to the right of Chi and unwinds DNA to the left of Chi; segments of unwound single-stranded DNA are sometimes, but not always, degraded before synapsis with homologous DNA.}, }
@article {pmid2556327, year = {1989}, author = {Thaler, DS and Sampson, E and Siddiqi, I and Rosenberg, SM and Thomason, LC and Stahl, FW and Stahl, MM}, title = {Recombination of bacteriophage lambda in recD mutants of Escherichia coli.}, journal = {Genome}, volume = {31}, number = {1}, pages = {53-67}, doi = {10.1139/g89-013}, pmid = {2556327}, issn = {0831-2796}, support = {5-T32-GM07413/GM/NIGMS NIH HHS/United States ; GM 33677/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacterial Proteins/genetics ; Bacteriophage lambda/*genetics ; Centrifugation, Density Gradient ; DNA Replication/genetics ; Deoxyribonucleases, Type II Site-Specific/genetics ; Escherichia coli/*genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*genetics ; Genotype ; Isotope Labeling ; Mutation ; Recombination, Genetic/*genetics ; Virus Replication/genetics ; }, abstract = {RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.}, }
@article {pmid2973573, year = {1988}, author = {Shpakovski, GV and Akhrem, AA and Berlin YuA, }, title = {Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure.}, journal = {Nucleic acids research}, volume = {16}, number = {21}, pages = {10199-10212}, pmid = {2973573}, issn = {0305-1048}, mesh = {Bacteriophage lambda/*genetics ; Base Sequence ; *Chromosome Deletion ; DNA, Viral/*genetics ; Escherichia coli/*genetics ; *Genes, Viral ; Molecular Sequence Data ; Plasmids ; Restriction Mapping ; }, abstract = {In studying molecular mechanisms of specialised transduction, the lacI (E. coli)-Ea47 (lambda) DNA junction in transducing bacteriophage lambda plac 5 has been structurally elucidated, thus yielding the complete sequence of lambda plac 5 DNA including the lac5 substitution, a well-known segment of lambdoid vectors. The lambda plac5 DNA is shown to consist of 19368 bp (lambda left arm) + 3924 bp (lac5 substitution) + 25353 bp (lambda right arm), totally amounting to 48645 bp. The presence of the phage rho bL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be much longer than it was postulated earlier, coding for 224 C-terminal amino acid residues of lac repressor. Both the recombination studied in this paper and the earlier studied abnormal prophage excision (2, 3) occur near to Chi-like structures (chi*lacI and chi*lom, respectively). On the basis of the data obtained, a key role of the E. coli RecBCD system and Chi-like sequences in the formation of deletions in bacterial cells is suggested.}, }
@article {pmid2975616, year = {1988}, author = {Rosenberg, SM}, title = {Chain-bias of Escherichia coli Rec-mediated lambda patch recombinants is independent of the orientation of lambda cos.}, journal = {Genetics}, volume = {120}, number = {1}, pages = {7-21}, pmid = {2975616}, issn = {0016-6731}, support = {5-T32-GM07413/GM/NIGMS NIH HHS/United States ; GM33677/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*genetics ; Crosses, Genetic ; DNA, Viral/genetics ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/metabolism ; Nucleic Acid Heteroduplexes ; *Recombination, Genetic ; Species Specificity ; }, abstract = {Chi is a hotspot for homologous recombination mediated by the RecBCD (Rec) pathway of Escherichia coli. For Rec-mediated recombination of phage lambda, the orientation of lambda cos in the lambda chromosome dictates the direction of travel of RecBCD enzyme through DNA and dictates which orientation of Chi or Chi-like sequences will be active in stimulating recombination. I previously found that Rec-mediated lambda patch heteroduplexes, stimulated by Chi or not, are chain-biased; at the lambda P locus, recombinant information resides on the lambda r chain. This bias exists in the presence or absence of Chi sites. Reported herein is the finding that r-chain-bias at the P locus is independent of the orientation of lambda cos and thus also independent of the orientation of active Chi's or Chi-like sequences and of the direction of travel of RecBCD enzyme. These results disprove previously elaborated models in which a chain-specific nick at Chi initiates recombination, and imply that some other chain-distinguishing process is involved with recombination. Replication and transcription are candidates for such a process.}, }
@article {pmid2966143, year = {1988}, author = {Poteete, AR and Fenton, AC and Murphy, KC}, title = {Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions.}, journal = {Journal of bacteriology}, volume = {170}, number = {5}, pages = {2012-2021}, pmid = {2966143}, issn = {0021-9193}, support = {AI18234/AI/NIAID NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*genetics ; DNA Replication ; DNA, Viral/metabolism ; Escherichia coli/enzymology/*genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/antagonists &amp; inhibitors/*genetics/metabolism ; Gene Expression Regulation ; Genes, Bacterial ; Genes, Viral ; Plasmids ; Recombination, Genetic ; Salmonella Phages/*genetics ; Viral Proteins/biosynthesis/*genetics ; }, abstract = {Plasmids that express the bacteriophage lambda gam gene or the P22 abc2 gene (with and without abc1) at controllable levels were placed in Escherichia coli and tested for effects on the activity of RecBCD. Like Gam, Abc2 inhibited the ATP-dependent exonuclease activity of RecBCD, apparently not by binding to DNA. However, Abc2-mediated inhibition was partial, while Gam-mediated inhibition was complete. Both Abc2 and Gam inhibited host system-mediated homologous recombination in a Chi-containing interval in the chromosome of a hybrid lambda phage; Abc2 inhibited it more strongly than Gam. Gam but not Abc2 spared a phage T4 gene 2 mutant from restriction by RecBCD; Abc2 exhibited weak sparing activity in combination with Abc1 and substantial activity in combination with both Abc1 and P22 homologous recombination function Erf. Either Gam or the combination of the lambda recombination functions Exo and Bet was sufficient to induce a mode of plasmid replication that produced linear multimers. The combination of Abc2, Abc1, and Erf also exhibited this activity. However, Erf was inactive, both by itself and in combination with Abc1; Abc2 had weak activity. These results indicate that Gam and Abc2 modulate the activity of RecBCD in significantly different ways. In comparison with lambda Gam, P22 Abc2 has a weak effect on RecBCD nuclease activity but a strong effect on its recombination-promoting activity.}, }
@article {pmid2958631, year = {1987}, author = {Cheng, KC and Smith, GR}, title = {Cutting of chi-like sequences by the RecBCD enzyme of Escherichia coli.}, journal = {Journal of molecular biology}, volume = {194}, number = {4}, pages = {747-750}, doi = {10.1016/0022-2836(87)90252-x}, pmid = {2958631}, issn = {0022-2836}, support = {5 T32 GM-07187/GM/NIGMS NIH HHS/United States ; GM-31693/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/genetics ; Base Sequence ; DNA, Viral ; Escherichia coli/*enzymology/genetics ; *Escherichia coli Proteins ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/*metabolism ; *Recombination, Genetic ; }, abstract = {Chi, 5' G-C-T-G-G-T-G-G 3', stimulates coliphage lambda recombination mediated by the major recombination pathway of Escherichia coli, the RecBC pathway. Purified RecBCD enzyme makes a single strand endonuclease cleavage four to six nucleotides to the 3' side of the chi sequence. Three sequences similar to chi, 5' A-C-T-G-G-T-G-G 3', 5' G-T-T-G-G-T-G-G 3' and 5' G-C-T-A-G-T-G-G 3', have partial recombinational hotspot activity in genetic crosses. We report here that purified RecBCD enzyme preferentially cuts four nucleotides to the right of the two most active chi-like octamers. The degree of cutting correlated with the genetic activities of these sequences; this result indicates that these cleavages are essential to the genetic activity of chi and chi-like sequences.}, }
@article {pmid2949853, year = {1987}, author = {Rosenberg, SM}, title = {Chi-stimulated patches are heteroduplex, with recombinant information on the phage lambda r chain.}, journal = {Cell}, volume = {48}, number = {5}, pages = {855-865}, doi = {10.1016/0092-8674(87)90082-1}, pmid = {2949853}, issn = {0092-8674}, support = {5-T32-GM07413/GM/NIGMS NIH HHS/United States ; GM33677/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*genetics ; Base Sequence ; DNA, Viral/*genetics ; Escherichia coli/*genetics ; Nucleic Acid Heteroduplexes/*genetics ; *Recombination, Genetic ; }, abstract = {Generalized recombination in Escherichia coli is elevated near Chi sites. In vitro, RecBCD enzyme can nick Chi a few nucleotides 3' of the terminal GG of the Chi sequence (5'-GCTGGTGG). The simplest model in which this nick at Chi participates in Chi function predicts that in phage lambda, Chi-stimulated recombinants not crossed-over for flanking markers (patches) should be heteroduplex, with recombinant information on the lambda I chain. I report here that patches are heteroduplex, but that recombinant information occurs primarily on the lambda r chain. This result rules out the simplest model in which the nick at Chi promotes initiation of recombination, forces reconsideration of Chi's role in recombination, and bears on molecular models for Rec-mediated recombination.}, }
@article {pmid2951295, year = {1987}, author = {Ennis, DG and Amundsen, SK and Smith, GR}, title = {Genetic functions promoting homologous recombination in Escherichia coli: a study of inversions in phage lambda.}, journal = {Genetics}, volume = {115}, number = {1}, pages = {11-24}, pmid = {2951295}, issn = {0016-6731}, support = {5 T32 CA 09229/CA/NCI NIH HHS/United States ; GM 31693/GM/NIGMS NIH HHS/United States ; GM 32194/GM/NIGMS NIH HHS/United States ; }, mesh = {Bacteriophage lambda/*genetics ; Chromosome Inversion ; DNA, Viral/genetics ; Escherichia coli/*genetics ; Models, Genetic ; *Recombination, Genetic ; Repetitive Sequences, Nucleic Acid ; }, abstract = {We have studied homologous recombination in a derivative of phage lambda containing two 1.4-kb repeats in inverted orientation. Inversion of the intervening 2.5-kb segment occurred efficiently by the Escherichia coli RecBC pathway but markedly less efficiently by the lambda Red pathway or the E. coli RecE or RecF pathways. Inversion by the RecBCD pathway was stimulated by Chi sites located to the right of the invertible segment; this stimulation decreased exponentially by a factor of about 2 for each 2.2 kb between the invertible segment and the Chi site. In addition to RecA protein and RecBCD enzyme, inversion by the RecBC pathway required single-stranded DNA binding protein, DNA gyrase, DNA polymerase I and DNA ligase. Inversion appeared to occur either intra- or intermolecularly. These results are discussed in the framework of a current molecular model for the RecBC pathway of homologous recombination.}, }