Role for radA/sms in recombination intermediate processing in Escherichia coli

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.     

Replication arrest-stimulated recombination: Dependence on the RecA paralog, RadA/Sms and translesion polymerase, DinB

Difficulties in replication can lead to breakage of the fork. Recombinational reactions restore the integrity of the fork through strand-invasion of the broken chromosome with its sister. If this occurs in the context of repeated DNA sequences, genetic rearrangements can result. We have proposed that this process accounts for stimulation of chromosomal rearrangements by mutations in Escherichia coli's replicative DNA helicase, DnaB. At its permissive temperature for growth, a dnaB107 mutant is a 1000-fold more likely to experience a deletion of a 787bp tandem repeated segment inserted in the E. coli chromosome than is a wild-type strain. We have previously shown that enhanced deletion in a dnaB107 strain is reduced in recA, recB and recG102 (formerly known as radC102) derivatives. Here I show that this enhanced recombination is dependent on other factors: the RuvA Holliday junction helicase, the RecJ single-strand DNA exonuclease, the RadA/Sms RecA-paralog protein of unknown function and, surprisingly, the DinB translesion polymerase. The requirement for these factors in DnaB-stimulated rearrangements is much greater than that observed for recombinational events such as P1 transduction. This may be because strand invasion into the repeats limits the extent of heteroduplex DNA that can be formed in the initial stage of recombination. I propose that RadA, RecG and RuvAB are critically required to stabilize the strand-invasion intermediate and that DinB polymerase extends the invading 3' strand to aid in re-initiation. The role of DinB in bacteria may be analogous to translesion DNA polymerase eta in eukaryotes, recently shown to aid recombination.     

Roles of Ruva, Ruvc and Recg Gene Functions in Normal and DNA Damage-Inducible Replication of the Escherichia Coli Chromosome

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.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives 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 recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam 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.     

Growth-dependent DNA breakage and cell death in a gyrase mutant of Salmonella.

A class of gyrase mutants of Salmonella enterica mimics the properties of bacteria exposed to quinolones. These mutants suffer spontaneous DNA breakage during normal growth and depend on recombinational repair for viability. Unlike quinolone-treated bacteria, however, they do not show accumulation of cleavable gyrase-DNA complexes. In recA or recB mutant backgrounds, the temperature-sensitive (ts) allele gyrA208 causes rapid cell death at 43 degrees. Here, we isolated "suppressor-of-death" mutations, that is, secondary changes that allow a gyrA208 recB double mutant to survive a prolonged exposure to 43 degrees and subsequently to form colonies at 28 degrees. In most isolates, the secondary change was itself a ts mutation. Three ts alleles were mapped in genes coding for amino acyl tRNA synthetases (alaS, glnS, and lysS). Allele alaS216 completely abolished DNA breakage in a gyrA208 recA double mutant. Likewise, treating this mutant with chloramphenicol prevented death and DNA damage at 43 degrees. Additional suppressors of gyrA208 lethality include rpoB mutations and, surprisingly, icd mutations inactivating isocitrate dehydrogenase. We postulate that the primary effect of the gyrase alteration is to hamper replication fork movement. Inhibiting DNA replication under conditions of continuing macromolecular synthesis ("unbalanced growth") activates a mechanism that causes DNA breakage and cell death, reminiscent of "thymineless" lethality.     

Effect of rec mutations on viability and processing of DNA damaged by methylmethane sulfonate in xth nth nfo cells of Escherichia coli

The role of recombination genes in the processing of DNA damaged by methlymethane sulfonate (MMS) was examined in an xth nth nfo strain of Escherichia coli K-12. Introduction of a recQ mutation did not increase the cell's sensitivity to MMS treatment. The presence of recF, recJ or recN mutation slightly increased the cell's sensitivity to MMS treatment. The introduction of recA or recB mutation into the cells led to inviability. Taken together, we suggest that replication of DNA containing apurinic/apyrimidinic (AP) sites in vivo will lead to the formation of secondary lesions. The repair of these secondary lesions requires the function of recA and recB genes, but does not appear to require recF, recJ, recQ or recN genes.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.     

Suppression of Escherichia coli recF mutations by recA-linked srfA mutations.

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.     

Role of DnaB helicase in UV-induced illegitimate recombination in Escherichia coli

To study the involvement of DNA replication in UV-induced illegitimate recombination, we examined the effect of temperature-sensitive dnaB mutations on illegitimate recombination and found that the frequency of illegitimate recombination was reduced by an elongation-deficient mutation, dnaB14, but not by an initiation-deficient mutation, dnaB252. This result indicates that DNA replication is required for UV-induced illegitimate recombination. In addition, the dnaB14 mutation also affected spontaneous or UV-induced illegitimate recombination enhanced by the recQ mutation. Nucleotide sequence analyses of the recombination junctions showed that DnaB-mediated illegitimate recombination is short homology dependent. Previously, Michel et al. (B. Michel, S. Ehrlich, and M. Uzest, EMBO J. 16:430--438, 1997) showed that thermal treatment of the temperature-sensitive dnaB8 mutant induces double-stranded breaks, implying that induction of illegitimate recombination occurs. To explain the discrepancy between the observations, we propose a model for DnaB function, in which the dnaB mutations may exhibit two types of responses, early and late responses, for double-stranded break formation. In the early response, replication forks stall at damaged DNA, resulting in the formation of double-stranded breaks, and the dnaB14 mutation reduces the double-stranded breaks shortly after temperature shift-up. On the other hand, in the late response, the arrested replication forks mediated by the dnaB8 mutation may induce double-stranded breaks after prolonged incubation.     

Requirement for RecFOR-mediated recombination in priA mutant

Restart of arrested replication forks is an important process and PriA, the main Escherichia coli replication restart protein, is essential for viability under any condition that increases the frequency of fork arrest. In priA mutant, replication forks are arrested by spontaneously occurring roadblocks and blocked replication forks persist as a result of the defect in replication restart. In the present work, we analysed how recombination proteins contribute to the viability of the priA mutant. RecFOR-mediated homologous recombination occurs in a large fraction of priA mutant cells, indicating a frequent formation of DNA single strand gaps and their recombinational repair. This high level of homologous recombination renders the proteins that resolve Holliday junctions recombination intermediates essential for viability. When homologous recombination is blocked at early steps by recFOR or recA inactivation, exonuclease V-mediated DNA degradation is required for full viability of priA mutants, indicating that unrepaired gaps are broken and that DNA degradation of the broken DNA allows the formation of viable cells. Models for the formation of single strand DNA gaps consequently to a replication restart defect and for gap processing are proposed.     

Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair.

To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharomyces pombe RAD50 homologue. The deletion is sensitive to a range of DNA-damaging agents and shows dynamic epistatic interactions with other recombination-repair genes. We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow DNA replication. We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment. Interestingly, in rad50Delta cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination. We propose that Rad50, an SMC-like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair. In support of this, we found that Rad50 functions in the same pathway for the repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re-initiation of replication after replication fork collapse.     

Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12

The orf gene of bacteriophage lambda, 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 Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG 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.     

lon incompatibility associated with mutations causing SOS induction: null uvrD alleles induce an SOS response in Escherichia coli

The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.     

Genetic recombination destabilizes (CTG)n.(CAG)n repeats in E. coli

The expansion of trinucleotide repeats has been implicated in 17 neurological diseases to date. Factors leading to the instability of trinucleotide repeat sequences have thus been an area of intense interest. Certain genes involved in mismatch repair, recombination, nucleotide excision repair, and replication influence the instability of trinucleotide repeats in both Escherichia coli and yeast. Using a genetic assay for repeat deletion in E. coli, the effect of mutations in the recA, recB, and lexA genes on the rate of deletion of (CTG)n.(CAG)n repeats of varying lengths were examined. The results indicate that mutations in recA and recB, which decrease the rate of recombination, had a stabilizing effect on (CAG)n.(CTG)n repeats decreasing the high rates of deletion seen in recombination proficient cells. Thus, recombination proficiency correlates with high rates of genetic instability in triplet repeats. Induction of the SOS system, however, did not appear to play a significant role in repeat instability, nor did the presence of triplet repeats in cells turn on the SOS response. A model is suggested where deletion during exponential growth may result from attempts to restart replication when paused at triplet repeats.     

Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide

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 (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2).     

Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis.

Inhibition of deoxyribonucleic acid (DNA) synthesis in Escherichia coli by chemical inhibitors or by shifting cultures of temperature-sensitive elongation (dnaE and dnaB) or initiation (dnaA) mutants to nonpermissive conditions led to greatly increased synthesis of the enzyme ribonucleoside diphosphate reductase, which catalyzes the first reaction unique to the pathway leading to DNA replication. In contrast to the Gudas and Pardee proposed model for control of the synthesis of DNA repair enzymes, in which both DNA inhibition and DNA degradation are involved, DNA synthesis inhibition in recA, recB, recC, or lex strains results in increased synthesis of ribonucleotide reductase, which suggests that DNA degradation is not required. We propose that inhibition of DNA synthesis causes a cell to accumulate an unknown compound that stimulates the initiation of a new round of DNA replication, and that this same signal is used to induce ribonucleotide reductase synthesis.     

Requirement for homologous recombination functions for expression of the mutA mistranslator tRNA-induced mutator phenotype in Escherichia coli

Expression of the Escherichia coli mutA mutator phenotype requires recA, recB, recC, ruvA, and ruvC gene, but not recD, recF, recO, or recR genes. Thus, the recBCD-dependent homologous recombination system is a component of the signal pathway that activates an error-prone DNA polymerase in mutA cells.     

Phosphorylation of Rad55 on serines 2, 8, and 14 is required for efficient homologous recombination in the recovery of stalled replication forks

DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.     

Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the beta clamp

Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four-way junction. RuvABC-catalysed resolution of this Holliday junction causes chromosome double-strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability. In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively. In the dnaEts recB strain, RuvABC-dependent DSBs caused by the dnaEts mutation occurred at 37 degrees C or 42 degrees C, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ and the helicase function of PriA. In the dnaNts recB mutant, RuvABC-dependent DSB caused by the dnaNts mutation occurred only at semi-permissive temperature, 37 degrees C, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability. In contrast, the dnaNts mutation did not cause chromosome breakage at 42 degrees C, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable. We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.     

Requirement for Protein Synthesis in rec-Dependent Repair of Deoxyribonucleic Acid in Escherichia coli after Ultraviolet or X Irradiation

Deprivation of amino acids required for growth or treatment with chloramphenicol or puromycin after irradiation reduced the survival of Rec(+) cells of Escherichia coli K-12 which had been exposed to either ultraviolet (UV) or X radiation. In contrast, these treatments caused little or no reduction in the survival of irradiated recA or recB mutants. The effect of chloramphenicol on the survival of X-irradiated cells was correlated with an inhibition of repair of single-strand breaks in irradiated deoxyribonucleic acid (DNA), previously shown to be controlled by recA and recB. In UV-irradiated cells no effect of chloramphenicol was detected on the repair of single-strand discontinuities in DNA replicated from UV-damaged templates, a process controlled by recA but not by recB. From this we concluded that inhibiting protein synthesis in UV or X-irradiated cells may interfere with some biochemical step in repair dependent upon the recB gene. When irradiated Rec(+) cells were cultured for a sufficient period of time in minimal growth medium before chloramphenicol treatment their survival was no longer decreased by the drug. After X irradiation this occurred in less than one generation time of the unirradiated control cells. After UV irradiation it occurred more slowly and was only complete after several generation times of the unirradiated controls. These observations indicated that replication of the entire irradiated genome was probably not required for rec-dependent repair of X-irradiated cells, although it might be required for rec-dependent repair of UV-irradiated cells.