recF Mutations Research Articles

Structural and functional characterization of oligomeric states of proteins in RecFOR pathway

RecFOR pathway is the principal repair pathway for double strand break and single strand gap repair in Thermus thermophilus. RecF and RecR exist as monomer and dimer in solution, interestingly; they undergo condition-dependent dimerization and tetramerization, respectively during the DNA break repair. However, their importance in protein-protein and protein-DNA interactions remains elusive. In this study, the three-dimensional crystal structures of the wild type RecF and RecR proteins are determined. Thereafter, the structural information is used to mutate the interface residues to cysteine to stabilize the dimeric and tetrameric states of the RecF and RecR proteins, respectively. A comparative study for their interactions with other cognate proteins and ssDNA in native and SSB (single strand binding protein) bound states was performed. RecF or RecFR complex displays a negligible affinity towards ssDNA. Conversely, the RecF mutants and its complexes with wild type RecR showed affinity towards ssDNA, suggesting, distinct modes of interaction of RecF and RecFR complex for ssDNA binding. In the presence of RecO, the stabilized tetrameric RecR showed a lower binding affinity for ssDNA as compared to the SSB bound ssDNA, indicating the importance of tetrameric RecR in stabilizing the RecOR complex on the SSB coated ssDNA. This provides an insight into the reduction of the binding affinity of SSB proteins with the ssDNA, which in turn enhances the recruitment of RecA for strand exchange.

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB+ sbcC+ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3′-5′ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3′ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.

Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions.

The RadA/Sms protein is a RecA-related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double-strand break repair for YejH.

Recombination and Annealing Pathways Compete for Substrates in Making rrn Duplications in Salmonella enterica

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.

Tolerance ofEscherichia colito Fluoroquinolone Antibiotics Depends on Specific Components of the SOS Response Pathway

Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called "persisters," depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance.

RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases

We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this Luria–Bertani (LB)-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoBH447R and rpoBD444G prevent activation of the Prrn core promoter in rich medium, but only rpoBH447R also suppresses the auxotrophy of a relA spoT mutant (stringent-like phenotype). rpoCH113R suppresses the thermo-sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoCP451L prevent R-loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication–transcription collisions. In a RecF+ context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG.

Antibacterial effects of silver ions on growth of gram-negative bacteria and biofilm formation

The average minimal inhibiting concentrations (MICs) of AgNO3 for the gram-negative bacteria Escherichia coli K12, Serratia proteamaculans 94, and Serratia liquefaciens MG1 were found to be 0.075–0.3 μg/ml and, for Pseudomonas aeruginosa PAO1 and P. chlororaphis 449, this concentration was 0.15–0.3 μg/ml. The formation of a biofilm by Escherichia coli AB1157 and S. proteamaculans 94 was completely inhibited by 0.3 μg/ml of AgNO3 and, in the case of Pseudomonas aeruginosa PAO1, biofilm formation was inhibited by 0.6 μg/ml of AgNO3. Mutations in E. coli genes that encode global regulators of gene expression, such as sigma S and sigma N subunits of RNA polymerase, the catabolite repression protein CRP, and the Lon protease, had no marked effect on the sensitivity of cells to silver. Wild-type E. coli strains and strains deficient in excision repair (uvrA and uvrB), SOS repair, and recombination (recA, lexA, recBC, and recF mutants) did not differ in their sensitivity to silver. This suggests that the sensitivity of the bacteria to the silver was not associated with DNA lesions that could be repaired by these repair and recombination systems. E. coli mutant strains deficient in OmpF or OmpC porins were three to four times more resistant to silver ions than the wild-type strain. Experiments with the pME6863 plasmid, which bears the gene of N-acyl-homoserine lactonase AiiA, demonstrated that the quorum sensing (QS) regulation did not participate in controlling the sensitivity of S. proteamaculans 94 and P. chlororaphis 449 to silver. The same conclusion was drawn from a comparison of AgNO3 MICs of the S. liquefaciens wild-type strain and a mutant strain deficient in QS.

RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions

RecA is required for recombinational processes and cell survival following UV-induced DNA damage. recA433 is a historically important mutant allele that contains a single amino acid substitution (R243H). This mutation separates the recombination and survival functions of RecA. recA433 mutants remain proficient in recombination as measured by conjugation or transduction, but are hypersensitive to UV-induced DNA damage. The cellular functions carried out by RecA require either recF pathway proteins or recBC pathway proteins to initiate RecA-loading onto the appropriate DNA substrates. In this study, we characterized the ability of recA433 to carry out functions associated with either the recF pathway or recBC pathway. We show that several phenotypic deficiencies exhibited by recA433 mutants are similar to recF mutants but distinct from recBC mutants. In contrast to recBC mutants, recA433 and recF mutants fail to process or resume replication following disruption by UV-induced DNA damage. However, recA433 and recF mutants remain proficient in conjugational recombination and are resistant to formaldehyde-induced protein–DNA crosslinks, functions that are impaired in recBC mutants. The results are consistent with a model in which the recA433 mutation selectively impairs RecA functions associated with the RecF pathway, while retaining the ability to carry out RecBCD pathway-mediated functions. These results are discussed in the context of the recF and recBC pathways and the potential substrates utilized in each case.

Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following γ-radiation-induced damage to pMTa4 DNA in E. coli

Purpose: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to γ-radiation in vivo.Materials and methods: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and γ-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R−)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of γ-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored.Results: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R− condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of γ-radiation-induced ‘ladder’. The ‘ladder’ was not observed after repair incubation, UV-irradiation or γ-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised.Conclusions: Wild, recF and recA strains of E. coli could repair γ-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.

RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis

When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli.

Roles of Double-strand Breaks, Nicks, and Gaps in Stimulating Deletions of CTG·CAG Repeats by Intramolecular DNA Repair

A series of plasmids harboring CTG.CAG repeats with double-strand breaks (DSB), single-strand nicks, or single-strand gaps (15 or 30 nucleotides) within the repeat regions were used to determine their capacity to induce genetic instabilities. These plasmids were introduced into Escherichia coli in the presence of a second plasmid containing a sequence that could support homologous recombination repair between the two plasmids. The transfer of a point mutation from the second to the first plasmid was used to monitor homologous recombination (gene conversion). Only DSBs increased the overall genetic instability. This instability took place by intramolecular repair, which was not dependent on RuvA. Double-strand break-induced instabilities were partially stabilized by a mutation in recF. Gaps of 30 nt formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nt did not induce expansions or deletions. Formation of this deletion product required the CTG.CAG repeats to be present in the single-stranded region and was stimulated by E.coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the intramolecular repair-induced instabilities and the formation of the 30 nt deletion product.

UV-C radiation induced conformational relaxation of pMTa4 DNA in Escherichia coli may be the cause of single strand breaks

Purpose: The biological consequences of initial physicochemical events following exposure of DNA to germicidal (254 nm) ultraviolet C (UV-C) radiation are not fully understood despite progress that has been made. In particular the cause of UV-C induced single strand breaks is not known. This question has been addressed in the present investigation.Materials and methods: A plasmid construct, pMTa4, was exposed to UV-C in vitro as well as in vivo after transforming the plasmid into a repair proficient wild type and repair deficient, recF, mutant of E. coli. Following UV exposure in vivo, the plasmid was isolated under repair non-permissive and permissive conditions. The plasmid isolate and the pure super-coiled closed circular (CC) topological form of the plasmid were analyzed by agarose gel electrophoresis. The dependence of UV-C induced damage and conformational changes on the dose of radiation as well as on the duration of post-irradiation repair incubations was observed. The influence of UV-C on hyperchromic change and intercalation of ethidium bromide into plasmid DNA were also recorded.Results: UV-C exposure of pMTa4 DNA in vitro and in vivo induced dose dependent, but sparsely placed, single strand breaks (SSB). While the wild type (AB1157) E. coli was able to repair SSB nearly completely under repair permissive condition, the recF (JC9239) mutant failed to do so. A dose-dependent relaxation of super-structure of CC form of pMTa4 DNA concomitant with enhanced ethidium bromide intercalation into the plasmid DNA was observed.Conclusion: It is proposed that the conformational relaxation generated negative super-coiling strain on the DNA backbone of CC form of plasmid as well as exposed chemical bonds for hydrolytic cleavage. This might be the cause of the production of sparsely placed single strand breaks in pMTa4 upon exposure to low doses of UV-C.

UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli

The roles of UvrD and Rep DNA helicases of Escherichia coli are not yet fully understood. In particular, the reason for rep uvrD double mutant lethality remains obscure. We reported earlier that mutations in recF, recO or recR genes suppress the lethality of uvrD rep, and proposed that an essential activity common to UvrD and Rep is either to participate in the removal of toxic recombination intermediates or to favour the proper progression of replication. Here, we show that UvrD, but not Rep, directly prevents homologous recombination in vivo. In addition to RecFOR, we provide evidence that RecA contributes to toxicity in the rep uvrD mutant. In vitro, UvrD dismantles the RecA nucleoprotein filament, while Rep has only a marginal activity. We conclude that UvrD and Rep do not share a common activity that is essential in vivo: while Rep appears to act at the replication stage, UvrD plays a role of RecA nucleoprotein filament remover. This activity of UvrD is similar to that of the yeast Srs2 helicase.

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.

Effect of mutations for the ruvABC genes on recombination between direct DNA repairs in Escherichia coli strains carrying extended tandem duplication

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants for ruvABC genes. Homologous recombination in duplications of rec+ strains and in recBC sbcB, recQ and recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.

Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12.

Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants.

Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization

A deletion was engineered in the cloned recF gene by digestion with suitable restriction endonucleases and a tetracycline resistance gene cartridge was inserted. The mutation was subsequently transferred to the Azotobacter vinelandii chromosome by double cross-over under pressure of tetracycline selection. A recF recA mutant was also constructed in a similar manner. The mutations were found to be stable and mutation of the wild-type recF gene was confirmed by Southern blot hybridization. Both the mutants were UV sensitive and recombination deficient. Mutations in genes involved in nitrogen fixation in A. vinelandii are rather frequent and obtained comparatively easily despite of the presence of multiple identical chromosomes in A. vinelandii. It has been speculated that some kind of `homogenotization' process operates which is responsible for the `transmission' of mutation from one chromosome to all the chromosomes. This process is not affected by a mutation in recF or recA or in both recF and recA.

Construction of arecFdeletion mutant ofAzotobacter vinelandiiand its characterization

Abstract A deletion was engineered in the cloned recF gene by digestion with suitable restriction endonucleases and a tetracycline resistance gene cartridge was inserted. The mutation was subsequently transferred to the Azotobacter vinelandii chromosome by double cross-over under pressure of tetracycline selection. A recF recA mutant was also constructed in a similar manner. The mutations were found to be stable and mutation of the wild-type recF gene was confirmed by Southern blot hybridization. Both the mutants were UV sensitive and recombination deficient. Mutations in genes involved in nitrogen fixation in A. vinelandii are rather frequent and obtained comparatively easily despite of the presence of multiple identical chromosomes in A. vinelandii . It has been speculated that some kind of `homogenotization' process operates which is responsible for the `transmission' of mutation from one chromosome to all the chromosomes. This process is not affected by a mutation in recF or recA or in both recF and recA .

ATP Hydrolysis and DNA Binding by the Escherichia coli RecF Protein

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.

Involvement of recF, recO, and recR genes in UV-radiation mutagenesis of Escherichia coli.

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, and recR genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations in recF, 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 of recA2020, 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.