Genetic evidence for the requirement of RecA loading activity in SOS induction after UV irradiation in Escherichia coli

The SOS response in Escherichia coli results in the coordinately induced expression of more than 40 genes which occurs when cells are treated with DNA-damaging agents. This response is dependent on RecA (coprotease), LexA (repressor), and the presence of single-stranded DNA (ssDNA). A prerequisite for SOS induction is the formation of a RecA-ssDNA filament. Depending on the DNA substrate, the RecA-ssDNA filament is produced by either RecBCD, RecFOR, or a hybrid recombination mechanism with specific enzyme activities, including helicase, exonuclease, and RecA loading. In this study we examined the role of RecA loading activity in SOS induction after UV irradiation. We performed a genetic analysis of SOS induction in strains with a mutation which eliminates RecA loading activity in the RecBCD enzyme (recB1080 allele). We found that RecA loading activity is essential for SOS induction. In the recB1080 mutant RecQ helicase is not important, whereas RecJ nuclease slightly decreases SOS induction after UV irradiation. In addition, we found that the recB1080 mutant exhibited constitutive expression of the SOS regulon. Surprisingly, this constitutive SOS expression was dependent on the RecJ protein but not on RecFOR, implying that there is a different mechanism of RecA loading for constitutive SOS expression.     

Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.     

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.     

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.     

RecJ nuclease is required for SOS induction after introduction of a double-strand break in a RecA loading deficient recB mutant of Escherichia coli

The SOS response is an important mechanism which allows Escherichia coli cells to maintain genome integrity. Two key proteins in SOS regulation are LexA (repressor) and RecA (coprotease). The signal for SOS induction is generated at the level of a RecA filament. Depending on the type of DNA damage, a RecA filament is produced by specific activities (helicase, nuclease and RecA loading) of either RecBCD, RecF or a hybrid recombination pathway. It was recently demonstrated that RecA loading activity is essential for the induction of the SOS response after UV-irradiation. In this paper we studied the genetic requirements for SOS induction after introduction of a double-strand break (DSB) by the I-SceI endonuclease in a RecA loading deficient recB mutant (recB1080). We monitored SOS induction by assaying beta-galactosidase activity and compared induction of the response between strains having one or more inactivated mechanisms of RecA loading and their derivatives. We found that simultaneous inactivation of both RecA loading functions (in recB1080 recO double mutant) partially impairs SOS induction after introduction of a DSB. However, we found that the RecJ nuclease is essential for SOS induction after the introduction of a DSB in the recB1080 mutant. This result indicates that RecJ is needed to prepare ssDNA for subsequent loading of RecA protein. It implies that an additional type of RecA loading could exist in the cell.     

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.     

UvrD controls the access of recombination proteins to blocked replication forks

Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA.     

Nucleolytic degradation of 3′-ending overhangs is essential for DNA-end resection in RecA-loading deficient recB mutants of Escherichia coli

Degradation of a 5′-ending strand is the hallmark of the universal process of DNA double strand break (DSB) resection, which results in creation of the central recombination intermediate, a 3′-ending overhang. Here we show that in Escherichia coli recB1080/recB1067 mutants, which are devoid of RecBCD's nuclease and RecA loading activities, degradation of the unwound 3′ tail is as essential as is degradation of its 5′-ending complement. Namely, a synergistic action of ExoI, ExoVII, SbcCD and ExoX single-strand specific exonucleases (ssExos) of 3′-5′ polarity was essential for preserving cell viability, DNA repair and homologous recombination in the recB1080/recB1067 mutants, to the same extent as the redundant action of 5′-tail trimming ssExos RecJ and ExoVII. recB1080 derivatives lacking 3′-5′ ssExos also showed a strong induction of the SOS response and greatly increased SOS-dependent mutagenesis. Furthermore, we show that ExoI and ExoVII ssExos act synergistically in suppressing illegitimate recombination in the recB1080 mutant but not in a wt strain, while working in concert with the RecQ helicase. Remarkably, 3′-5′ ssExos show synergism with RecQ helicase in the recB1080 mutant in all the assays tested. The effect of inactivation of 3′-5′ ssExos in the recB1080/recB1067 mutants was much stronger than in wt, recD, and recB strains. These results demonstrate that the presence of a long, reactive 3′ overhang can be as toxic for a cell as its complete absence, i.e. it may prevent DSB repair. Our results indicate that coupling of helicase and RecA-loading activity during dsDNA-end resection is crucial in avoiding the deleterious effects of a long and stabile 3′ tail in E. coli.     

DNA end resection controls the balance between homologous and illegitimate recombination in Escherichia coli

Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD's RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3' tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3' ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

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.     

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.     

RecQ-dependent death-by-recombination in cells lacking RecG and UvrD

Maintenance of genomic stability is critical for all cells. Homologous recombination (HR) pathways promote genome stability using evolutionarily conserved proteins such as RecA, SSB, and RecQ, the Escherichia coli homologue of five human proteins at least three of which suppress genome instability and cancer. A previous report indicated that RecQ promotes the net accumulation in cells of intermolecular HR intermediates (IRIs), a net effect opposite that of the yeast and two human RecQ homologues. Here we extend those conclusions. We demonstrate that cells that lack both UvrD, an inhibitor of RecA-mediated strand exchange, and RecG, a DNA helicase implicated in IRI resolution, are inviable. We show that the uvrD recG cells die a “death-by-recombination” in which IRIs accumulate blocking chromosome segregation. First, their death requires RecA HR protein. Second, the death is accompanied by cytogenetically visible failure to segregate chromosomes. Third, FISH analyses show that the unsegregated chromosomes have completed replication, supporting the hypothesis that unresolved IRIs prevented the segregation. Fourth, we show that RecQ and induction of the SOS response are required for the accumulation of replicated, unsegregated chromosomes and death, as are RecF, RecO, and RecJ. ExoI exonuclease and MutL mismatch-repair protein are partially required. This set of genes is similar but not identical to those that promote death-by-recombination of ΔuvrD Δruv cells. The data support models in which RecQ promotes the net accumulation in cells of IRIs and RecG promotes resolution of IRIs that form via pathways not wholly identical to those that produce the IRIs resolved by RuvABC. This implies that RecG resolves intermediates other than or in addition to standard Holliday junctions resolved by RuvABC. The role of RecQ in net accumulation of IRIs may be shared by one or more of its human homologues.     

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.     

Effects of recJ, recQ, and recFOR mutations on recombination in nuclease-deficient recB recD double mutants of Escherichia coli

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.     

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.     

Thymineless death is inhibited by CsrA in Escherichia coli lacking the SOS response

Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind⁻) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA⁻ SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells.     

Requirements for ATP binding and hydrolysis in RecA function in Escherichia coli

RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli . ATP hydrolysis is known to be important for RecA's roles in recombination and DNA repair. In vitro reactions modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo . The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA–GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA⁺, depending on the ratio of the two proteins. RecA4159 was unable to enter RecA⁺ filaments on DNA or storage structures and was recessive to RecA⁺. It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament.     

The Escherichia coli UvrD helicase is essential for Tus removal during recombination-dependent replication restart from Ter sites

Blocking replication forks in the Escherichia coli chromosome by ectopic Ter sites renders the RecBCD pathway of homologous recombination and SOS induction essential for viability. In this work, we show that the E. coli helicase II (UvrD) is also essential for the growth of cells where replication forks are arrested at ectopic Ter sites. We propose that UvrD is required for Tus removal from Ter sites. The viability of a SOS non-inducible Ter-blocked strain is fully restored by the expression of the two SOS-induced proteins UvrD and RecA at high level, indicating that these are the only two SOS-induced proteins required for replication across Ter/Tus complexes. Several observations suggest that UvrD acts in concert with homologous recombination and we propose that UvrD is associated with recombination-initiated replication forks and that it removes Tus when a PriA-dependent, restarted replication fork goes across the Ter/Tus complex. Finally, expression of the UvrD homologue from Bacilus subtilis PcrA restores the growth of uvrD-deficient Ter-blocked cells, indicating that the capacity to dislodge Tus is conserved in this distant bacterial species.     

Survival and induction of SOS in Escherichia coli treated with cisplatin, UV-irradiation, or mitomycin C are dependent on the function of the RecBC and RecFOR pathways of homologous recombination

Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The antitumor effects of these drugs are due to the formation of bifunctional adducts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study the cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilitate the production of a molecular signal for induction of the SOS response. Strains used in the SOS chromotest have a fusion of lacZ with the sfiA (sulA) gene so that the amount of SOS inducing signal, which is modulated by the ability of the cell to repair DNA, is measured by assaying beta-galactosidase activity. SOS induction in a strain proficient in homologous recombination (HR) was compared with that in isogenic strains deficient in HR due to a blocked RecBC pathway caused by a recB mutation or a blocked RecFOR pathway caused by a recO mutation. The effect of cisplatin treatment in a uvrA mutant strain blocked at the first step of NER was compared with that in an isogenic strain proficient in NER. Cellular resistance was measured as percent colony forming units (cfu) for cells treated with increasing doses of cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demonstrated by decreased survival in mutants with the recB mutant being more sensitive than the recO mutant. SOS induction levels were elevated in the sensitive recB strain relative to the HR proficient strain possibly due to stalled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act at daughter strand gaps in newly synthesized DNA to mediate production of the signal for SOS induction. Proficiency in NER was necessary for both survival and high levels of SOS induction in cisplatin treated cells.