Roles of RecJ,RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli

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.     

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.     

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.     

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli . Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD  ::Tn 5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD -mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA , recF , recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD ⁺ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.     

Antipairing and strand transferase activities of E. coli helicase II (UvrD).

The product of the uvrD gene of Escherichia coli, UvrD (helicase II), is known to be involved in methyl-directed mismatch repair, transposon excision and uvrABC excision repair. In conjugational crosses, various uvrD mutants have been reported to result in higher, lower or unaffected recombination frequencies. In an attempt to clarify the role of UvrD in recombination, we have studied in vitro its effects on two key reactions driven by RecA, homologous pairing and strand exchange. We show here that UvrD efficiently prevents or reverses RecA-mediated homologous pairing. Unexpectedly, we also found that it can stimulate RecA-driven branch migration and even catalyze strand exchange in the absence of RecA. A possible in vivo role for these antagonistic activities is discussed.     

An in vitro complementation assay for the Escherichia coli uvrD gene product.

An in vitro assay specific for the product of the uvrD gene of Escherichia coli has been developed. This assay, derived from properties of uvrD mutants revealed by in vivo experiments, is based on the necessity for a functional UvrD protein for complete rejoining of covalently closed circular DNA during the excision repair of UV-induced damage. Extracts prepared from gently lysed uvrD101 mutant cells are capable of restoring UV-damaged DNA to its covalently closed circular form when provided with a functional UvrD protein from other repair-deficient cell extracts or from partially purified protein fractions. This assay was employed to monitor the activity of the UvrD protein after several steps of fractionation. The partially purified UvrD protein does not complement extracts deficient in DNA polymerase I or temperature-sensitive in DNA ligase; it does, however, complement extracts from strains mutant at the uvrE and recL loci, which are considered allelic with the uvrD locus.     

UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli

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.     

UvrD2 is essential in Mycobacterium tuberculosis, but its helicase activity is not required

UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosis has two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2 gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2 is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2 at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.     

UvrD limits the number and intensities of RecA-green fluorescent protein structures in Escherichia coli K-12

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.     

Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli

We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism.     

Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.     

RecF recombination pathway in Escherichia coli cells lacking RecQ, UvrD and HelD helicases

In recBCD sbcB sbcC(D) mutants of Escherichia coli homologous recombination proceeds via RecF pathway, which is thought to require RecQ, UvrD and HelD helicases at its initial stage. It was previously suggested that depletion of all three helicases totally abolishes the RecF pathway. The present study (re)examines the roles of these helicases in transductional recombination, and in recombinational repair of UV-induced DNA damage in the RecF pathway. The study has employed the ΔrecBCD ΔsbcB sbcC201 and ΔrecBCD sbcB15 sbcC201 strains, carrying combinations of mutations in recQ, uvrD, and helD genes. We show that in ΔrecBCD ΔsbcB sbcC201 strains, recombination requires exclusively the RecQ helicase. In ΔrecBCD sbcB15 sbcC201 strains, RecQ may be partially substituted by UvrD helicase. The HelD helicase is dispensable for recombination in both backgrounds. Our results also suggest that significant portion of recombination events in the RecF pathway is independent of RecQ, UvrD and HelD. These events are initiated either by RecJ nuclease alone or by RecJ nuclease associated with an unknown helicase. Inactivation of exonuclease VII by a xseA mutation further decreases the requirement for helicase activity in the RecF pathway. We suggest that elimination of nucleases acting on 3' single-strand DNA ends reduces the necessity for helicases in initiation of recombination.     

Characterization of DNA helicase II from a uvrD252 mutant of Escherichia coli.

The loss of DNA helicase II (UvrD) in Escherichia coli results in sensitivity to UV light and increased levels of spontaneous mutagenesis. While the effects of various uvrD alleles have been analyzed in vivo, the proteins produced by these alleles have not been examined in any detail. We have cloned one of these alleles, uvrD252, and determined the site of the mutation conferring the phenotype. In addition, the protein it encodes has been purified to homogeneity and characterized in vitro. The mutation responsible for the phenotype was identified as a glycine-to-aspartic-acid change in the putative ATP-binding domain. In comparison to wild-type DNA helicase II, the UvrD252 enzyme exhibited reduced levels of ATPase activity and a large increase in the Km for ATP. The ability of UvrD252 to unwind DNA containing single-stranded regions, as well as DNA containing only nicks, was reduced in comparison to that of the wild-type enzyme. Possible interpretations of these results in relation to the phenotypes of the uvrD252 mutant are discussed. This represents the first detailed analysis of the biochemical properties of a mutant DNA helicase II protein.     

Essential bacterial helicases that counteract the toxicity of recombination proteins

PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation. Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant. We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent.     

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.     

Role of the recJ gene product in UV-induced illegitimate recombination at the hotspot.

Illegitimate recombination between a prophage and adjacent bacterial DNA is the first step in the formation of specialized transducing phage. Such recombination is rare, but it is greatly enhanced by UV irradiation. We studied the mechanism of UV-induced illegitimate recombination by examining the effect of rec mutations on the frequency of lambda bio transducing phage and found that an Escherichia coli recJ mutation reduces it by 3- to 10-fold. In addition, the recombination hotspot, which accounts for approximately 60% of lambda bio transducing phages in wild-type bacteria, was not detected in the recJ mutant. Introduction of a RecJ overexpression plasmid into the recJ mutant recovered the recombination at the hotspot. These results indicate that the RecJ protein preferentially stimulates illegitimate recombination at the hotspot. Both the hotspot and the non- hotspot sites have short regions of homology, but only the hotspot sites contain common direct-repeat sequences. We propose a model based on the 5'-3' exonuclease activity of RecJ to explain the involvement of this protein in illegitimate recombination at the hotspot.     

Important role for Mycobacterium tuberculosis UvrD1 in pathogenesis and persistence apart from its function in nucleotide excision repair

Mycobacterium tuberculosis survives and replicates in macrophages, where it is exposed to reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the roles of UvrA and UvrD1, thought to be parts of the nucleotide excision repair pathway of M. tuberculosis. Strains in which uvrD1 was inactivated either alone or in conjunction with uvrA were constructed. Inactivation of uvrD1 resulted in a small colony phenotype, although growth in liquid culture was not significantly affected. The sensitivity of the mutant strains to UV irradiation and to mitomycin C highlighted the importance of the targeted genes for nucleotide excision repair. The mutant strains all exhibited heightened susceptibility to representatives of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). The uvrD1 and the uvrA uvrD1 mutants showed decreased intracellular multiplication following infection of macrophages. Most importantly, the uvrA uvrD1 mutant was markedly attenuated following infection of mice by either the aerosol or the intravenous route.     

Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype.

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we isolated temperature-sensitive gyrA mutants that confer spontaneous illegitimate recombination and spontaneous induction of lambda prophage at higher frequencies than that in the wild-type. After reconstruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyrAI203V+gyrAI205V, show the same properties as those described above. With respect to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of recombination junctions of lambdabio transducing phages formed spontaneously in these mutants showed that the parental E. coli bio and lambda recombination sites have a homologous sequence of only 0. 7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysis of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrAL488P mutations contain amino acid substitutions of Leu492-->Pro and Leu488-->Pro, respectively, which correspond to the alpha18 helix in the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gyrAI205V mutations contain Ile203-->Val and Ile205-->Val, respectively, which correspond to the alpha10' helix, also in the breakage-reunion domain of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 protein that contains the L492P mutation has an apparently normal supercoiling activity, but it also produces a small amount of linear DNA in the absence of DNA gyrase inhibitor during the supercoiling reaction, suggesting that the mutant DNA gyrase may have a defect at the step of religation or a defect in the subunit interaction. These results suggest that the recombination is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two alpha helices, alpha10' and alpha18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA.     

DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair.

DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.