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1
Luisi-DeLuca, C., S. T. Lovett, and R. D. Kolodner. "Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants." Genetics 122, no. 2 (June 1, 1989): 269–78. http://dx.doi.org/10.1093/genetics/122.2.269.
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Abstract The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.
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Stohl, Elizabeth A., and H. Steven Seifert. "Neisseria gonorrhoeae DNA Recombination and Repair Enzymes Protect against Oxidative Damage Caused by Hydrogen Peroxide." Journal of Bacteriology 188, no. 21 (August 25, 2006): 7645–51. http://dx.doi.org/10.1128/jb.00801-06.
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ABSTRACT The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H2O2) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H2O2 damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H2O2. Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H2O2 resistance as well. Finally, we show that RecA protein levels did not increase as a result of H2O2 treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H2O2 resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H2O2.
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Lovett, S. T., C. Luisi-DeLuca, and R. D. Kolodner. "The genetic dependence of recombination in recD mutants of Escherichia coli." Genetics 120, no. 1 (September 1, 1988): 37–45. http://dx.doi.org/10.1093/genetics/120.1.37.
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Abstract RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.
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Marinus, M. G. "Recombination Is Essential for Viability of anEscherichia coli dam (DNA Adenine Methyltransferase) Mutant." Journal of Bacteriology 182, no. 2 (January 15, 2000): 463–68. http://dx.doi.org/10.1128/jb.182.2.463-468.2000.
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ABSTRACT Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, orruvC could not be constructed, whereas damderivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA(Ind−) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recAand ruvAB need to be derepressed to allow fordam mutant survival. The presence of mutS ormutL mutations allowed the construction of dam lexA (Ind−) derivatives. The requirement forrecA, recB, recC, ruvA,ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability ofdam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.
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Sawitzke, J. A., and F. W. Stahl. "Phage lambda has an analog of Escherichia coli recO, recR and recF genes." Genetics 130, no. 1 (January 1, 1992): 7–16. http://dx.doi.org/10.1093/genetics/130.1.7.
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Abstract The RecF pathway catalyzes generalized recombination in Escherichia coli that is mutant for recBC, sbcB and sbcC. This pathway operating on conjugational recombination requires the recA, recF, recJ, recN, recO, recQ, recR, ruvA, ruvB and ruvC genes. In contrast, lambda mutant for its own recombination genes, int, red alpha and red beta, requires only the recA and recJ genes to recombine efficiently in recBC sbcB sbcC cells. Deletion of an open reading frame in the ninR region of lambda results in an additional requirement for recO, recR and recF in order to recombine in recBC sbcB sbcC mutant cells. This function, designated orf for recO-, recR- and recF-like function, is largely RecF pathway specific.
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Shiraishi, Kouya, Katsuhiro Hanada, Yoichiro Iwakura, and Hideo Ikeda. "Roles of RecJ, RecO, and RecR in RecET-Mediated Illegitimate Recombination in Escherichia coli." Journal of Bacteriology 184, no. 17 (September 1, 2002): 4715–21. http://dx.doi.org/10.1128/jb.184.17.4715-4721.2002.
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ABSTRACT We analyzed effects of overexpression of RecE and RecT on illegitimate recombination during prophage induction in Escherichia coli and found that frequencies of spontaneous and UV-induced illegitimate recombination are enhanced by coexpression of RecE and RecT in the wild type, but the enhanced recombination was reduced by recJ, recO, or recR mutation. The results indicated that RecET-mediated illegitimate recombination depends on the functions of RecJ, RecO, and RecR, suggesting that the RecE and RecJ exonucleases play different roles in this recombination pathway and that the RecO and RecR proteins also play important roles in the recombination. On the other hand, the frequency of the RecET-mediated illegitimate recombination was enhanced by a recQ mutation, implying that the RecQ protein plays a role in suppression of RecET-mediated illegitimate recombination. It was also found that RecET-mediated illegitimate recombination is independent of the RecA function with UV irradiation, but it is enhanced by the recA mutation without UV irradiation. Based on these results, we propose a model for the roles of RecJOR on RecET-mediated illegitimate recombination.
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Beam, Cynthia E., Catherine J. Saveson, and Susan T. Lovett. "Role for radA/sms in Recombination Intermediate Processing in Escherichia coli." Journal of Bacteriology 184, no. 24 (December 15, 2002): 6836–44. http://dx.doi.org/10.1128/jb.184.24.6836-6844.2002.
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ABSTRACT RadA/Sms is a highly conserved eubacterial protein that shares sequence similarity with both RecA strand transferase and Lon protease. We examined mutations in the radA/sms gene of Escherichia coli for effects on conjugational recombination and sensitivity to DNA-damaging agents, including UV irradiation, methyl methanesulfonate (MMS), mitomycin C, phleomycin, hydrogen peroxide, and hydroxyurea (HU). Null mutants of radA were modestly sensitive to the DNA-methylating agent MMS and to the DNA strand breakage agent phleomycin, with conjugational recombination decreased two- to threefold. We combined a radA mutation with other mutations in recombination genes, including recA, recB, recG, recJ, recQ, ruvA, and ruvC. A radA mutation was strongly synergistic with the recG Holliday junction helicase mutation, producing profound sensitivity to all DNA-damaging agents tested. Lesser synergy was noted between a mutation in radA and recJ, recQ, ruvA, ruvC, and recA for sensitivity to various genotoxins. For survival after peroxide and HU exposure, a radA mutation surprisingly suppressed the sensitivity of recA and recB mutants, suggesting that RadA may convert some forms of damage into lethal intermediates in the absence of these functions. Loss of radA enhanced the conjugational recombination deficiency conferred by mutations in Holliday junction-processing function genes, recG, ruvA, and ruvC. A radA recG ruv triple mutant had severe recombinational defects, to the low level exhibited by recA mutants. These results establish a role for RadA/Sms in recombination and recombinational repair, most likely involving the stabilization or processing of branched DNA molecules or blocked replication forks because of its genetic redundancy with RecG and RuvABC.
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Romero, Zachary J., Stefanie H. Chen, Thomas Armstrong, Elizabeth A. Wood, Antoine van Oijen, Andrew Robinson, and Michael M. Cox. "Resolving Toxic DNA repair intermediates in every E. coli replication cycle: critical roles for RecG, Uup and RadD." Nucleic Acids Research 48, no. 15 (July 9, 2020): 8445–60. http://dx.doi.org/10.1093/nar/gkaa579.
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Abstract DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.
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Mendonca, V. M., and S. W. Matson. "Genetic analysis of delta helD and delta uvrD mutations in combination with other genes in the RecF recombination pathway in Escherichia coli: suppression of a ruvB mutation by a uvrD deletion." Genetics 141, no. 2 (October 1, 1995): 443–52. http://dx.doi.org/10.1093/genetics/141.2.443.
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Abstract Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background, we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD ant the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and regN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Surprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the deltauvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two "RecF" homologous recombination pathways operating in a recBCsbcB(C) strain background.
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Ivančić-Baće, Ivana, Erika Salaj-Šmic, and Krunoslav Brčić-Kostić. "Effects of recJ, recQ, and recFOR Mutations on Recombination in Nuclease-Deficient recB recD Double Mutants of Escherichia coli." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1350–56. http://dx.doi.org/10.1128/jb.187.4.1350-1356.2005.
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ABSTRACT The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5′→3′ exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5′→3′ exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5′→3′ exonuclease is an element of the RecF recombination machinery.
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Ivančić-Baće, Ivana, Petra Peharec, Sunčana Moslavac, Nikolina Škrobot, Erika Salaj-Šmic†, and Krunoslav Brčić-Kostić. "RecFOR Function Is Required for DNA Repair and Recombination in a RecA Loading-Deficient recB Mutant of Escherichia coli." Genetics 163, no. 2 (February 1, 2003): 485–94. http://dx.doi.org/10.1093/genetics/163.2.485.
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Abstract The RecA loading activity of the RecBCD enzyme, together with its helicase and 5′ → 3′ exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF– background the recB1080 mutant is recombination deficient, whereas in a recF+ genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D–) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5′ → 3′ exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.
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Belle, Jerilyn J., Andrew Casey, Charmain T. Courcelle, and Justin Courcelle. "Inactivation of the DnaB Helicase Leads to the Collapse and Degradation of the Replication Fork: a Comparison to UV-Induced Arrest." Journal of Bacteriology 189, no. 15 (May 25, 2007): 5452–62. http://dx.doi.org/10.1128/jb.00408-07.
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ABSTRACT Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including recF, recO, recR, recJ, recG, and xonA. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.
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Friedman-Ohana, Rachel, Iris Karunker, and Amikam Cohen. "A RecG-Independent Nonconservative Branch Migration Mechanism in Escherichia coli Recombination." Journal of Bacteriology 181, no. 23 (December 1, 1999): 7199–205. http://dx.doi.org/10.1128/jb.181.23.7199-7205.1999.
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ABSTRACT To gain insight regarding the mechanisms that extend heteroduplex joints in Escherichia coli recombination, we investigated the effect of recG and ruv genotypes on heteroduplex strand polarity in intramolecular recombination products. We also examined the cumulative effect of mutational inactivation of RecG and single-strand-specific exonucleases on recombination proficiency and the role of Chi sites in RecG-independent recombination. All four strands of the two homologs were incorporated into heteroduplex structures in wild-type cells and in ruvmutants. However, in recG mutants heteroduplexes were generated almost exclusively by pairing the invasive 3′-ending strand with its complementary strand. To explain the dependence of strand exchange reciprocity on RecG activity, we propose that alternative mechanisms may extend the heteroduplex joints after homologous pairing: a reciprocal RecG-mediated mechanism and a nonreciprocal mechanism, mediated by RecA and single-strand-specific exonucleases. The cumulative effect of recG and recJ orxonA mutations on recombination proficiency and the inhibitory effect of recJ and xonA activities on heteroduplex formation by the 5′-ending strands are consistent with this proposal.
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Sanchez, Humberto, Dawit Kidane, M. Castillo Cozar, Peter L. Graumann, and Juan C. Alonso. "Recruitment of Bacillus subtilis RecN to DNA Double-Strand Breaks in the Absence of DNA End Processing." Journal of Bacteriology 188, no. 2 (January 15, 2006): 353–60. http://dx.doi.org/10.1128/jb.188.2.353-360.2006.
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ABSTRACT The recognition and processing of double-strand breaks (DSBs) to a 3′ single-stranded DNA (ssDNA) overhang structure in Bacillus subtilis is poorly understood. Mutations in addA and addB or null mutations in recJ (ΔrecJ), recQ (ΔrecQ), or recS (ΔrecS) genes, when present in otherwise-Rec+ cells, render cells moderately sensitive to the killing action of different DNA-damaging agents. Inactivation of a RecQ-like helicase (ΔrecQ or ΔrecS) in addAB cells showed an additive effect; however, when ΔrecJ was combined with addAB, a strong synergistic effect was observed with a survival rate similar to that of ΔrecA cells. RecF was nonepistatic with RecJ or AddAB. After induction of DSBs, RecN-yellow fluorescent protein (YFP) foci were formed in addAB ΔrecJ cells. AddAB and RecJ were required for the formation of a single RecN focus, because in their absence multiple RecN-YFP foci accumulated within the cells. Green fluorescent protein-RecA failed to form filamentous structures (termed threads) in addAB ΔrecJ cells. We propose that RecN is one of the first recombination proteins detected as a discrete focus in live cells in response to DSBs and that either AddAB or RecQ(S)-RecJ are required for the generation of a duplex with a 3′-ssDNA tail needed for filament formation of RecA.
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Ryder, L., M. C. Whitby, and R. G. Lloyd. "Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli." Journal of Bacteriology 176, no. 6 (1994): 1570–77. http://dx.doi.org/10.1128/jb.176.6.1570-1577.1994.
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Poteete, Anthony R. "Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function in Escherichia coli K-12." Journal of Bacteriology 186, no. 9 (May 1, 2004): 2699–707. http://dx.doi.org/10.1128/jb.186.9.2699-2707.2004.
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ABSTRACT The orf gene of bacteriophage λ, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Δ(recC ptr recB recD)::P tac gam bet exo pae cI ΔrecG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.
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Centore, Richard C., and Steven J. Sandler. "UvrD Limits the Number and Intensities of RecA-Green Fluorescent Protein Structures in Escherichia coli K-12." Journal of Bacteriology 189, no. 7 (January 26, 2007): 2915–20. http://dx.doi.org/10.1128/jb.01777-06.
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ABSTRACT RecA is important for recombination, DNA repair, and SOS induction. In Escherichia coli, RecBCD, RecFOR, and RecJQ prepare DNA substrates onto which RecA binds. UvrD is a 3′-to-5′ helicase that participates in methyl-directed mismatch repair and nucleotide excision repair. uvrD deletion mutants are sensitive to UV irradiation, hypermutable, and hyper-rec. In vitro, UvrD can dissociate RecA from single-stranded DNA. Other experiments suggest that UvrD removes RecA from DNA where it promotes unproductive reactions. To test if UvrD limits the number and/or the size of RecA-DNA structures in vivo, an uvrD mutation was combined with recA-gfp. This recA allele allows the number of RecA structures and the amount of RecA at these structures to be assayed in living cells. uvrD mutants show a threefold increase in the number of RecA-GFP foci, and these foci are, on average, nearly twofold higher in relative intensity. The increased number of RecA-green fluorescent protein foci in the uvrD mutant is dependent on recF, recO, recR, recJ, and recQ. The increase in average relative intensity is dependent on recO and recQ. These data support an in vivo role for UvrD in removing RecA from the DNA.
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Pandya, Gagan A., In-Young Yang, Arthur P. Grollman, and Masaaki Moriya. "Escherichia coli Responses to a Single DNA Adduct." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6598–604. http://dx.doi.org/10.1128/jb.182.23.6598-6604.2000.
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ABSTRACT To study the mechanisms by which Escherichia colimodulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1,N 6-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF,recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.
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Asai, T., and T. Kogoma. "Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of the Escherichia coli chromosome." Genetics 137, no. 4 (August 1, 1994): 895–902. http://dx.doi.org/10.1093/genetics/137.4.895.
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Abstract Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR). We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination. By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination. Continuation of iSDR, on the other hand, is inhibited by these mutations. The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement. Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants. We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions.
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Nakayama, Koji, Susumu Shiota, and Hiroaki Nakayama. "Thymineless death in Escherichia coli mutants deficient in the RecF recombination pathway." Canadian Journal of Microbiology 34, no. 7 (July 1, 1988): 905–7. http://dx.doi.org/10.1139/m88-157.
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Like recF and recQ mutants studied earlier, two other classes of Escherichia coli mutants defective in the RecF conjugal recombination pathway, recJ and recO, were found to be partially resistant to thymineless death. In contrast, a recN mutant, also belonging to the pathway, was indistinguishable from the wild type with respect to thymineless death.
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Ren, Li, Abu Amar M. Al Mamun, and M. Zafri Humayun. "Requirement for Homologous Recombination Functions for Expression of the mutA Mistranslator tRNA-Induced Mutator Phenotype in Escherichia coli." Journal of Bacteriology 182, no. 5 (March 1, 2000): 1427–31. http://dx.doi.org/10.1128/jb.182.5.1427-1431.2000.
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ABSTRACT Expression of the Escherichia coli mutA mutator phenotype requires recA, recB,recC, ruvA, and ruvC gene, but notrecD, recF, recO, orrecR genes. Thus, the recBCD-dependent homologous recombination system is a component of the signal pathway that activates an error-prone DNA polymerase inmutA cells.
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Fernández, Silvia, Alexei Sorokin, and Juan C. Alonso. "Genetic Recombination in Bacillus subtilis 168: Effects of recU and recSMutations on DNA Repair and Homologous Recombination." Journal of Bacteriology 180, no. 13 (July 1, 1998): 3405–9. http://dx.doi.org/10.1128/jb.180.13.3405-3409.1998.
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ABSTRACT Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions. TheyppB:cat and ypbC:catnull alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec+ B. subtilis strain. The yppBgene complemented the defect of the recG40 strain.yppB and ypbC and their respective null alleles were termed “recU” and “recU1” (recU:cat) and “recS” and “recS1” (recS:cat), respectively. The recU and recS mutations were introduced into rec-deficient strains representative of the α (recF), β (addA5 addB72), γ (recH342), and ɛ (recG40) epistatic groups. The recU mutation did not modify the sensitivity ofrecH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells. TherecS mutation did not modify the sensitivity ofaddAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recUcells. The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells. Intramolecular recombination was blocked inrecS recF, recS addAB, and recS recU cells. RecU and RecS have no functional counterparts inEscherichia coli. Altogether, these data indicate that therecU and recS proteins are required for DNA repair and intramolecular recombination and that the recF(α epistatic group), addAB (β), recH (γ),recU (ɛ), and recS genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed recS within a new group, termed “ζ.”
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Maisnier-Patin, Sophie, Kurt Nordström, and Santanu Dasgupta. "RecA-Mediated Rescue of Escherichia coli Strains with Replication Forks Arrested at the Terminus." Journal of Bacteriology 183, no. 20 (October 15, 2001): 6065–73. http://dx.doi.org/10.1128/jb.183.20.6065-6073.2001.
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ABSTRACT The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1 CW) or counterclockwise (intR1 CC) orientations. Only theintR1 CC strain, with replication forks arrested at the terminus, required RecA for survival. Unlike the strains with RecA-dependent replication known so far, theintR1 CC strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability. The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC andintR1 CC strains. In theintR1 CC strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus. This was consistent with suppression of the RecA dependence of theintR1 CC strain by inactivating Tus, the protein needed to block replication forks at Ter sites. Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways.
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Liu, Ying-Hsiu, Ann-Joy Cheng, and Tzu-chien V. Wang. "Involvement of recF, recO, and recR Genes in UV-Radiation Mutagenesis ofEscherichia coli." Journal of Bacteriology 180, no. 7 (April 1, 1998): 1766–70. http://dx.doi.org/10.1128/jb.180.7.1766-1770.1998.
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ABSTRACT The recF, recO, and recR genes were originally identified as those affecting the RecF pathway of recombination in Escherichia coli cells. Several lines of evidence suggest that the recF, recO, andrecR genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations inrecF, recO, or recR greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp− (trpE65) to a Trp+phenotypes. Introduction of the defective lexA51 mutation [lexA51(Def)] and/or UmuD′ into recF,recO, and recR mutants failed to restore normal UVM in the mutants. On the other hand, the presence ofrecA2020, a suppressor mutation for recF,recO, and recR mutations, restored normal UVM in recF, recO, and recR mutants. These results indicate an involvement of the recF,recO, and recR genes and their products in UVM, possibly by affecting the third role of RecA in UVM.
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Foster, Patricia L., and William A. Rosche. "Increased Episomal Replication Accounts for the High Rate of Adaptive Mutation in recD Mutants of Escherichia coli." Genetics 152, no. 1 (May 1, 1999): 15–30. http://dx.doi.org/10.1093/genetics/152.1.15.
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Abstract Adaptive mutation has been studied extensively in FC40, a strain of Escherichia coli that cannot metabolize lactose (Lac-) because of a frameshift mutation affecting the lacZ gene on its episome. recD mutants of FC40, in which the exonuclease activity of RecBCD (ExoV) is abolished but its helicase activity is retained, have an increased rate of adaptive mutation. The results presented here show that, in several respects, adaptive mutation to Lac+ involves different mechanisms in recD mutant cells than in wild-type cells. About half of the apparent increase in the adaptive mutation rate of recD mutant cells is due to a RecA-dependent increase in episomal copy number and to growth of the Lac- cells on the lactose plates. The remaining increase appears to be due to continued replication of the episome, with the extra copies being degraded or passed to recD+ recipients. In addition, the increase in adaptive mutation rate in recD mutant cells is (i) dependent on activities of the single-stranded exonucleases, RecJ and ExoI, which are not required for (in fact, slightly inhibit) adaptive mutation in wild-type cells, and (ii) enhanced by RecG, which opposes adaptive mutation in wild-type cells.
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Niga, T., H. Yoshida, H. Hattori, S. Nakamura, and H. Ito. "Cloning and sequencing of a novel gene (recG) that affects the quinolone susceptibility of Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 41, no. 8 (August 1997): 1770–74. http://dx.doi.org/10.1128/aac.41.8.1770.
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In a study of the quinolone resistance genes in Staphylococcus aureus, a recG homolog was cloned as a gene affecting quinolone susceptibility. Sequencing analysis revealed that the gene consists of 2,061 nucleotides and encodes a 686-amino-acid polypeptide, which shows 38, 39, and 50% amino acid identity with the RecGs of Escherichia coli, Haemophilus influenzae, and Streptococcus pneumoniae, respectively. Seven helicase motifs are well conserved in the gene product. A plasmid carrying the gene complemented a recG-deficient mutant of E. coli with respect to mitomycin hypersusceptibility, demonstrating that the gene product is functionally equivalent to E. coli RecG. These results indicate that the gene is the recG gene of S. aureus. S. aureus RCM101 (recG::Tn551), designated S. aureus 3f33, is four to eight times more susceptible to quinolones than the parent strain, RCM101. The transformation of strain 3f33 with a plasmid carrying the S. aureus recG gene made it as quinolone resistant as strain RCM101. These results suggest that the recG gene is involved in the repair of DNA damage resulting from quinolone treatment in S. aureus.
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Li, Shisheng, and Raymond Waters. "Escherichia coli Strains Lacking Protein HU Are UV Sensitive due to a Role for HU in Homologous Recombination." Journal of Bacteriology 180, no. 15 (August 1, 1998): 3750–56. http://dx.doi.org/10.1128/jb.180.15.3750-3756.1998.
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ABSTRACT hupA and hupB encode the α and β subunits of the Escherichia coli histone-like protein HU. Here we show that E. coli hup mutants are sensitive to UV in the rec+ sbc +, recBC sbcA, recBC sbcBC, umuDC,recF, and recD backgrounds. However,hupAB mutations do not enhance the UV sensitivity of resolvase-deficient recG ruvA strains. hupAB uvrA and hupAB recG strains are supersensitive to UV.hup mutations enhance the UV sensitivity ofruvA strains to a much lesser extent but enhance that ofrus-1 ruvA strains to the same extent as forrus+ ruv + strains. Our results suggest that HU plays a role in recombinational DNA repair that is not specifically limited to double-strand break repair or daughter strand gap repair; the lack of HU affects the RecG RusA and RuvABC pathways for Holliday junction processing equally if the two pathways are equally active in recombinational repair; the function of HU is not in the substrate processing step or in the RecFOR-directed synapsis action during recombinational repair. Furthermore, the UV sensitivity ofhup mutants cannot be suppressed by overexpression of wild-type or mutant gyrB, which confers novobiocin resistance, or by different concentrations of a gyrase inhibitor that can increase or decrease the supercoiling of chromosomal DNA.
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Lestini, Roxane, and Bénédicte Michel. "UvrD and UvrD252 Counteract RecQ, RecJ, and RecFOR in a rep Mutant of Escherichia coli." Journal of Bacteriology 190, no. 17 (June 20, 2008): 5995–6001. http://dx.doi.org/10.1128/jb.00620-08.
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ABSTRACT Rep and UvrD are two related Escherichia coli helicases, and inactivating both is lethal. Based on the observation that the synthetic lethality of rep and uvrD inactivation is suppressed in the absence of the recombination presynaptic proteins RecF, RecO, or RecR, it was proposed that UvrD is essential in the rep mutant to counteract a deleterious RecFOR-dependent RecA binding. We show here that the synthetic lethality of rep and uvrD mutations is also suppressed by recQ and recJ inactivation but not by rarA inactivation. Furthermore, it is independent of the action of UvrD in nucleotide excision repair and mismatch repair. These observations support the idea that UvrD counteracts a deleterious RecA binding to forks blocked in the rep mutant. An ATPase-deficient mutant of UvrD [uvrD(R284A)] is dominant negative in a rep mutant, but only in the presence of all RecQJFOR proteins, suggesting that the UvrD(R284A) mutant protein is deleterious when it counteracts one of these proteins. In contrast, the uvrD252 mutant (G30D), which exhibits a strongly decreased ATPase activity, is viable in a rep mutant, where it allows replication fork reversal. We conclude that the residual ATPase activity of UvrD252 prevents a negative effect on the viability of the rep mutant and allows UvrD to counteract the action of RecQ, RecJ, and RecFOR at forks blocked in the rep mutant. Models for the action of UvrD at blocked forks are proposed.
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Foster, Patricia L., Jeffrey M. Trimarchi, and Russell A. Maurer. "Two Enzymes, Both of Which Process Recombination Intermediates, Have Opposite Effects on Adaptive Mutation in Escherichia coli." Genetics 142, no. 1 (January 1, 1996): 25–37. http://dx.doi.org/10.1093/genetics/142.1.25.
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Reversion of a lac – frameshift allele carried on an F′ episome in Escherichia coli occurs at a high rate when the cells are placed under lactose selection. Unlike Lac+ mutations that arise during nonselective growth, the production of these adaptive mutations requires the RecA-RecBCD pathway for recombination. In this report, we show that enzymes that process recombination intermediates are involved in the mutagenic process. RuvAB and RecG, E. coli’s two enzymes for translocating Holliday junctions, have opposite effects: RuvAB is required for RecA-dependent adaptive mutations, whereas RecG inhibits them.
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Lloyd, R. G., and C. Buckman. "Conjugational recombination in Escherichia coli: genetic analysis of recombinant formation in Hfr x F- crosses." Genetics 139, no. 3 (March 1, 1995): 1123–48. http://dx.doi.org/10.1093/genetics/139.3.1123.
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Abstract The formation of recombinants during conjugation between Hfr and F- strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located approximately 500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located > 300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange(s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec+ crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to approximately 1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.
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Carrasco, Begoña, M. Castillo Cozar, Rudi Lurz, Juan C. Alonso, and Silvia Ayora. "Genetic Recombination in Bacillus subtilis 168: Contribution of Holliday Junction Processing Functions in Chromosome Segregation." Journal of Bacteriology 186, no. 17 (September 1, 2004): 5557–66. http://dx.doi.org/10.1128/jb.186.17.5557-5566.2004.
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ABSTRACT Bacillus subtilis mutants classified within the ε (ruvA, ΔruvB, ΔrecU, and recD) and η (ΔrecG) epistatic groups, in an otherwise rec+ background, render cells impaired in chromosomal segregation. A less-pronounced segregation defect in ΔrecA and Δsms (ΔradA) cells was observed. The repair deficiency of addAB, ΔrecO, ΔrecR, recH, ΔrecS, and ΔsubA cells did not correlate with a chromosomal segregation defect. The sensitivity of ε epistatic group mutants to DNA-damaging agents correlates with ongoing DNA replication at the time of exposure to the agents. The Δsms (ΔradA) and ΔsubA mutations partially suppress the DNA repair defect in ruvA and recD cells and the segregation defect in ruvA and ΔrecG cells. The Δsms (ΔradA) and ΔsubA mutations partially suppress the DNA repair defect of ΔrecU cells but do not suppress the segregation defect in these cells. The ΔrecA mutation suppresses the segregation defect but does not suppress the DNA repair defect in ΔrecU cells. These results result suggest that (i) the RuvAB and RecG branch migrating DNA helicases, the RecU Holliday junction (HJ) resolvase, and RecD bias HJ resolution towards noncrossovers and that (ii) Sms (RadA) and SubA proteins might play a role in the stabilization and or processing of HJ intermediates.
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Vlašić, Ignacija, Ramona Mertens, Elena M. Seco, Begoña Carrasco, Silvia Ayora, Günther Reitz, Fabian M. Commichau, Juan C. Alonso, and Ralf Moeller. "Bacillus subtilis RecA and its accessory factors, RecF, RecO, RecR and RecX, are required for spore resistance to DNA double-strand break." Nucleic Acids Research 42, no. 4 (November 26, 2013): 2295–307. http://dx.doi.org/10.1093/nar/gkt1194.
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Abstract Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions generates a replication stress to germinating spores, and the RecA·ssDNA filament might act by preventing potentially dangerous forms of DNA repair occurring during replication. RecA might stabilize a stalled fork or prevent or promote dissolution of reversed forks rather than its cleavage that should require end-processing.
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Gupta, Richa, Stewart Shuman, and Michael S. Glickman. "RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria." Journal of Bacteriology 197, no. 19 (July 20, 2015): 3121–32. http://dx.doi.org/10.1128/jb.00290-15.
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ABSTRACTMycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation ofadnABorrecOindividually causes partial impairment of HR, but loss ofadnABandrecOin combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNAin vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis ofrecFandrecRin mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCEThis study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284–2295, 2013,http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.
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Kidane, Dawit, and Peter L. Graumann. "Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells." Journal of Cell Biology 170, no. 3 (August 1, 2005): 357–66. http://dx.doi.org/10.1083/jcb.200412090.
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We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced ∼30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 μm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions.
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Thaler, David S., Elizabeth Sampson, Imran Siddiqi, Susan M. Rosenberg, Lynn C. Thomason, Franklin W. Stahl, and Mary M. Stahl. "Recombination of bacteriophage λ in recD mutants of Escherichia coli." Genome 31, no. 1 (January 1, 1989): 53–67. http://dx.doi.org/10.1139/g89-013.
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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 λ is strikingly altered by recD null mutations in the host. When λ DNA replication is blocked, recombination in recD mutant strains is high near λ's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along λ unless the λ chromosome contains a χ sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on λ. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent χ). The distribution of exchanges in recD mutants is strongly affected by λ DNA replication. The distribution of exchanges on λ growing in rec+ cells is not influenced by DNA replication. The exchange distribution along λ in recD mutant cells is independent of χ in a variety of conditions. Recombination in rec+ cells is χ 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 λ 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 λ'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 χ sequence.Key words: χ sequence, RecBCD pathway, Red pathway, RecBC‡ pathway.
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Lombardo, Mary-Jane, and Susan M. Rosenberg. "radC102 of Escherichia coliIs an Allele of recG." Journal of Bacteriology 182, no. 22 (November 15, 2000): 6287–91. http://dx.doi.org/10.1128/jb.182.22.6287-6291.2000.
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ABSTRACT The radC102 mutation causes mild UV and X-ray sensitivity and was mapped previously to near pyrE andrecG at 82 min on the Escherichia colichromosome (I. Felzenszwalb, N. J. Sargentini, and K. C. Smith, Radiat. Res. 97:615–625, 1984). We report thatradC102 has two striking phenotypes characteristic ofrecG mutations. First, it causes dramatically increased RecA-dependent mutation in a stationary-phase mutation assay. Second, it causes extreme UV sensitivity in combination with ruvmutations affecting the RuvABC Holliday junction resolution system. DNA sequencing of the radC and recG genes inradC102 strains revealed that the radC102mutation creates a stop codon in recG that is predicted to truncate the RecG protein at 410 of 603 amino acids. A low-copy-number plasmid carrying the radC + gene did not affect the UV sensitivity of a wild-type strain, a radC102 strain, or arecG258::Tn10mini-kanstrain. We conclude that radC102 is an allele ofrecG and that the function of the RadC protein remains to be determined.
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Kang, Josephine, and Martin J. Blaser. "Repair and Antirepair DNA Helicases in Helicobacter pylori." Journal of Bacteriology 190, no. 12 (March 28, 2008): 4218–24. http://dx.doi.org/10.1128/jb.01848-07.
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ABSTRACT Orthologs of RecG and RuvABC are highly conserved among prokaryotes; in Escherichia coli, they participate in independent pathways that branch migrate Holliday junctions during recombinational DNA repair. RecG also has been shown to directly convert stalled replication forks into Holliday junctions. The bacterium Helicobacter pylori, with remarkably high levels of recombination, possesses RecG and RuvABC homologs, but in contrast to E. coli, H. pylori RecG limits recombinational repair. We now show that the RuvABC pathway plays the prominent, if not exclusive, repair role. By introducing an E. coli resolvase (RusA) into H. pylori, the repair and recombination phenotypes of the ruvB mutant but not the recG mutant were improved. Our results indicate that RecG and RuvB compete for Holliday junction structures in recombinational repair, but since a classic RecG resolvase is absent from H. pylori, deployment of the RecG pathway is lethal. We propose that evolutionary loss of the H. pylori RecG resolvase provides an “antirepair” pathway allowing for selection of varied strains. Such competition between repair and antirepair provides a novel mechanism to maximize fitness at a bacterial population level.
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Warren, Garrett, Richard Stein, Hassane Mchaourab, and Brandt Eichman. "Movement of the RecG Motor Domain upon DNA Binding Is Required for Efficient Fork Reversal." International Journal of Molecular Sciences 19, no. 10 (October 6, 2018): 3049. http://dx.doi.org/10.3390/ijms19103049.
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RecG catalyzes reversal of stalled replication forks in response to replication stress in bacteria. The protein contains a fork recognition (“wedge”) domain that binds branched DNA and a superfamily II (SF2) ATPase motor that drives translocation on double-stranded (ds)DNA. The mechanism by which the wedge and motor domains collaborate to catalyze fork reversal in RecG and analogous eukaryotic fork remodelers is unknown. Here, we used electron paramagnetic resonance (EPR) spectroscopy to probe conformational changes between the wedge and ATPase domains in response to fork DNA binding by Thermotoga maritima RecG. Upon binding DNA, the ATPase-C lobe moves away from both the wedge and ATPase-N domains. This conformational change is consistent with a model of RecG fully engaged with a DNA fork substrate constructed from a crystal structure of RecG bound to a DNA junction together with recent cryo-electron microscopy (EM) structures of chromatin remodelers in complex with dsDNA. We show by mutational analysis that a conserved loop within the translocation in RecG (TRG) motif that was unstructured in the RecG crystal structure is essential for fork reversal and DNA-dependent conformational changes. Together, this work helps provide a more coherent model of fork binding and remodeling by RecG and related eukaryotic enzymes.
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Kang, Josephine, Don Tavakoli, Ariane Tschumi, Rahul A. Aras, and Martin J. Blaser. "Effect of Host Species on RecG Phenotypes in Helicobacter pylori and Escherichia coli." Journal of Bacteriology 186, no. 22 (November 15, 2004): 7704–13. http://dx.doi.org/10.1128/jb.186.22.7704-7713.2004.
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ABSTRACT Recombination is a fundamental mechanism for the generation of genetic variation. Helicobacter pylori strains have different frequencies of intragenomic recombination, arising from deletions and duplications between DNA repeat sequences, as well as intergenomic recombination, facilitated by their natural competence. We identified a gene, hp1523, that influences recombination frequencies in this highly diverse bacterium and demonstrate its importance in maintaining genomic integrity by limiting recombination events. HP1523 shows homology to RecG, an ATP-dependent helicase that in Escherichia coli allows repair of damaged replication forks to proceed without recourse to potentially mutagenic recombination. Cross-species studies done show that hp1523 can complement E. coli recG mutants in trans to the same extent as E. coli recG can, indicating that hp1523 has recG function. The E. coli recG gene only partially complements the hp1523 mutation in H. pylori. Unlike other recG homologs, hp1523 is not involved in DNA repair in H. pylori, although it has the ability to repair DNA when expressed in E. coli. Therefore, host context appears critical in defining the function of recG. The fact that in E. coli recG phenotypes are not constant in other species indicates the diverse roles for conserved recombination genes in prokaryotic evolution.
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Torres, Rubén, Carolina Gándara, Begoña Carrasco, Ignacio Baquedano, Silvia Ayora, and Juan C. Alonso. "DisA Limits RecG Activities at Stalled or Reversed Replication Forks." Cells 10, no. 6 (May 31, 2021): 1357. http://dx.doi.org/10.3390/cells10061357.
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The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c-di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.
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Hong, X., G. W. Cadwell, and T. Kogoma. "Escherichia coli RecG and RecA proteins in R-loop formation." EMBO Journal 14, no. 10 (May 1995): 2385–92. http://dx.doi.org/10.1002/j.1460-2075.1995.tb07233.x.
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Saveson, Catherine J., and Susan T. Lovett. "Tandem Repeat Recombination Induced by Replication Fork Defects in Escherichia coli Requires a Novel Factor, RadC." Genetics 152, no. 1 (May 1, 1999): 5–13. http://dx.doi.org/10.1093/genetics/152.1.5.
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Abstract DnaB is the helicase associated with the DNA polymerase III replication fork in Escherichia coli. Previously we observed that the dnaB107(ts) mutation, at its permissive temperature, greatly stimulated deletion events at chromosomal tandem repeats. This stimulation required recA, which suggests a recombinational mechanism. In this article we examine the genetic dependence of recombination stimulated by the dnaB107 mutation. Gap repair genes recF, recO, and recR were not required. Mutations in recB, required for double-strand break repair, and in ruvC, the Holliday junction resolvase gene, were synthetically lethal with dnaB107, causing enhanced temperature sensitivity. The hyperdeletion phenotype of dnaB107 was semidominant, and in dnaB107/dnaB+ heterozygotes recB was partially required for enhanced deletion, whereas ruvC was not. We believe that dnaB107 causes the stalling of replication forks, which may become broken and require repair. Misalignment of repeated sequences during RecBCD-mediated repair may account for most, but not all, of deletion stimulated by dnaB107. To our surprise, the radC gene, like recA, was required for virtually all recombination stimulated by dnaB107. The biochemical function of RadC is unknown, but is reported to be required for growth-medium-dependent repair of DNA strand breaks. Our results suggest that RadC functions specifically in recombinational repair that is associated with the replication fork.
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Handa, Naofumi, Asao Ichige, and Ichizo Kobayashi. "Contribution of RecFOR machinery of homologous recombination to cell survival after loss of a restriction–modification gene complex." Microbiology 155, no. 7 (July 1, 2009): 2320–32. http://dx.doi.org/10.1099/mic.0.026401-0.
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Loss of a type II restriction–modification (RM) gene complex, such as EcoRI, from a bacterial cell leads to death of its descendent cells through attack by residual restriction enzymes on undermethylated target sites of newly synthesized chromosomes. Through such post-segregational host killing, these gene complexes impose their maintenance on their host cells. This finding led to the rediscovery of type II RM systems as selfish mobile elements. The host prokaryote cells were found to cope with such attacks through a variety of means. The RecBCD pathway of homologous recombination in Escherichia coli repairs the lethal lesions on the chromosome, whilst it destroys restricted non-self DNA. recBCD homologues, however, appear very limited in distribution among bacterial genomes, whereas homologues of the RecFOR proteins, responsible for another pathway, are widespread in eubacteria, just like the RM systems. In the present work, therefore, we examined the possible contribution of the RecFOR pathway to cell survival after loss of an RM gene complex. A recF mutation reduced survival in an otherwise rec-positive background and, more severely, in a recBC sbcBC background. We also found that its effect is prominent in the presence of specific non-null mutant forms of the RecBCD enzyme: the resistance to killing seen with recC1002, recC1004, recC2145 and recB2154 is severely reduced to the level of a null recBC allele when combined with a recF, recO or recR mutant allele. Such resistance was also dependent on RecJ and RecQ functions. UV resistance of these non-null recBCD mutants is also reduced by recF, recJ or recQ mutation. These results demonstrate that the RecFOR pathway of recombination can contribute greatly to resistance to RM-mediated host killing, depending on the genetic background.
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Saxena, Sarika, Satoru Nagatoishi, Daisuke Miyoshi, and Naoki Sugimoto. "Structural and Functional Characterization of RecG Helicase under Dilute and Molecular Crowding Conditions." Journal of Nucleic Acids 2012 (2012): 1–8. http://dx.doi.org/10.1155/2012/392039.
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In an ATP-dependent reaction, theEscherichia coliRecG helicase unwinds DNA junctionsin vitro. We present evidence of a unique protein conformational change in the RecG helicase from anα-helix to aβ-strand upon an ATP binding under dilute conditions using circular dichroism (CD) spectroscopy. In contrast, under molecular crowding conditions, theα-helical conformation was stable even upon an ATP binding. These distinct conformational behaviors were observed to be independent of Na+and Mg2+. Interestingly, CD measurements demonstrated that the spectra of a frayed duplex decreased with increasing of the RecG concentration both under dilute and molecular crowding conditions in the presence of ATP, suggesting that RecG unwound the frayed duplex. Our findings raise the possibility that theα-helix andβ-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively.
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Fonville, Natalie C., Matthew D. Blankschien, Daniel B. Magner, and Susan M. Rosenberg. "RecQ-dependent death-by-recombination in cells lacking RecG and UvrD." DNA Repair 9, no. 4 (April 2010): 403–13. http://dx.doi.org/10.1016/j.dnarep.2009.12.019.
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Morimatsu, Katsumi, Yun Wu, and Stephen C. Kowalczykowski. "RecFOR Proteins Target RecA Protein to a DNA Gap with Either DNA or RNA at the 5′ Terminus." Journal of Biological Chemistry 287, no. 42 (August 17, 2012): 35621–30. http://dx.doi.org/10.1074/jbc.m112.397034.
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The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1–2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000–2000 nucleotides downstream (5′ → 3′) of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5′-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks.
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Poteete, Anthony R., Anita C. Fenton, and Kenan C. Murphy. "Roles of RuvC and RecG in Phage λ Red-Mediated Recombination." Journal of Bacteriology 181, no. 17 (September 1, 1999): 5402–8. http://dx.doi.org/10.1128/jb.181.17.5402-5408.1999.
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ABSTRACT The recombination properties of Escherichia colistrains expressing the red genes of bacteriophage λ and lacking recBCD function either by mutation or by expression of λ gam were examined. The substrates for recombination were nonreplicating λ chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in ruvC+recG+ , ruvC recG +,ruvC+ recG, and ruvC recG hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome’s restriction site marker. In this case, recombinant formation was found to be partially dependent upon ruvC function, especially in arecG mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the cat gene, with flankinglac sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher inrecG than in recG +. These observations favor models in which RecG tends to push invading 3′-ended strands back out of recombination intermediates.
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Donaldson, Janet R., Charmain T. Courcelle, and Justin Courcelle. "RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli." Genetics 166, no. 4 (April 1, 2004): 1631–40. http://dx.doi.org/10.1093/genetics/166.4.1631.
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Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.
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Laufer, C. S., J. B. Hays, B. E. Windle, T. S. Schaefer, E. H. Lee, S. L. Hays, and M. R. McClure. "Enhancement of Escherichia coli plasmid and chromosomal recombination by the Ref function of bacteriophage P1." Genetics 123, no. 3 (November 1, 1989): 465–76. http://dx.doi.org/10.1093/genetics/123.3.465.
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Abstract The Ref activity of phage P1 enhances recombination between two defective lacZ genes in the Escherichia coli chromosome (lac- x lac- recombination). Plasmid recombination, both lac- x lac- and tet- x tet-, was measured by transformation of recA strains, and was also assayed by measurement of beta-galactosidase. The intracellular presence of recombinant plasmids was verified directly by Southern blotting. Ref stimulated recombination of plasmids in rec+ and rec(BCD) cells by 3-6-fold, and also the low level plasmid recombination in recF cells. RecA-independent plasmid recombination, either very low level (recA cells) or high level (recB recC sbcA recA cells), was not stimulated. Ref stimulated both intramolecular and intermolecular plasmid recombination. Both normal and Ref-stimulated lac- x lac- chromosomal recombination, expected to be mostly RecBC-dependent in wild-type bacteria, were affected very little by a recF mutation. We have previously reported Ref stimulation of lac- x lac- recombination in recBC sbcB bacteria, a process known to be RecF-dependent. Chromosomal recombination processes thought to involve activated recombination substrates, e.g., Hfr conjugation, P1 transduction, were not elevated by Ref activity. We hypothesize that Ref acts by unknown mechanisms to activate plasmid and chromosomal DNA for RecA-mediated recombination, and that the structures formed are substrates for both RecF-dependent (plasmid, chromosomal) and Rec(BCD)-dependent (chromosomal) recombination pathways.
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Castellanos, Mildred, and David Romero. "The Extent of Migration of the Holliday Junction Is a Crucial Factor for Gene Conversion in Rhizobium etli." Journal of Bacteriology 191, no. 15 (June 5, 2009): 4987–95. http://dx.doi.org/10.1128/jb.00111-09.
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ABSTRACT Gene conversion, defined as the nonreciprocal transfer of DNA, is one result of homologous recombination. Three steps in recombination could give rise to gene conversion: (i) DNA synthesis for repair of the degraded segment, (ii) Holliday junction migration, leading to heteroduplex formation, and (iii) repair of mismatches in the heteroduplex. There are at least three proteins (RuvAB, RecG, and RadA) that participate in the second step. Their roles have been studied for homologous recombination, but evidence of their relative role in gene conversion is lacking. In this work, we showed the effect on gene conversion of mutations in ruvB, recG, and radA in Rhizobium etli, either alone or in combination, using a cointegration strategy previously developed in our laboratory. The results indicate that the RuvAB system is highly efficient for gene conversion, since its absence provokes smaller gene conversion segments than those in the wild type as well as a shift in the preferred position of conversion tracts. The RecG system possesses a dual role for gene conversion. Inactivation of recG leads to longer gene conversion tracts than those in the wild type, indicating that its activity may hinder heteroduplex extension. However, under circumstances where it is the only migration activity present (as in the ruvB radA double mutant), conversion segments can still be seen, indicating that RecG can also promote gene conversion. RadA is the least efficient system in R. etli but is still needed for the production of detectable gene conversion tracts.