Spontaneous and double-strand break repair-associated quasipalindrome and frameshift mutagenesis in budding yeast: role of mismatch repair.

Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1000 times greater than during replication. Many mutations involve dissociating a partially- copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MATα, repaired by an HMR::KI-URA3 donor. We inserted into HMR::KI-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield MAT::KI-ura3::QP + 4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP + 4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP + 4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (poI3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a + 1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

Generation and Repair of Postreplication Gaps in Escherichia coli.

When replication forks encounter template lesions, one result is lesion skipping, where the stalled DNA polymerase transiently stalls, disengages, and then reinitiates downstream to leave the lesion behind in a postreplication gap. Despite considerable attention in the 6 decades since postreplication gaps were discovered, the mechanisms by which postreplication gaps are generated and repaired remain highly enigmatic. This review focuses on postreplication gap generation and repair in the bacterium Escherichia coli. New information to address the frequency and mechanism of gap generation and new mechanisms for their resolution are described. There are a few instances where the formation of postreplication gaps appears to be programmed into particular genomic locations, where they are triggered by novel genomic elements.

Characterization of the Escherichia coli XPD/Rad3 iron-sulfur helicase YoaA in complex with the DNA polymerase III clamp loader subunit chi (χ).

Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described. Here, we characterize YoaA and show that purified YoaA contains iron. YoaA and χ form a complex that is stable through three chromatographic steps, including gel filtration chromatography. When overexpressed in the absence of χ, YoaA is mostly insoluble. In addition, we show the YoaA-χ complex has DNA-dependent ATPase activity. Our measurement of the YoaA-χ helicase activity illustrates for the first time YoaA-χ translocates on ssDNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ssDNA gap, for DNA/DNA unwinding. Furthermore, YoaA-χ preferentially unwinds forked duplex DNA that contains both 3' and 5' single-stranded overhangs versus duplex DNA with only a 5' overhang. Finally, we demonstrate YoaA-χ can unwind damaged DNA that contains an abasic site or damage on 3' ends that stall replication extension. These results are the first biochemical evidence demonstrating YoaA is a bona fide iron-sulfur helicase, and we further propose the physiologically relevant form of the helicase is YoaA-χ.

DnaA and SspA regulation of the iraD gene of Escherichia coli: an alternative DNA damage response independent of LexA/RecA.

The transcription factor RpoS of Escherichia coli controls many genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by azidothymidine. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp and are regulated by regulatory inactivation of DnaA, dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays, we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced regulatory inactivation of DnaA. We also demonstrate that the RNA-polymerase associated factor, stringent starvation protein A, induced by the accumulation of ppGpp, also affects iraD expression, with a positive effect on constitutive expression and a negative effect on azidothymidine-induced expression.

Genetic Analysis of DinG Family Helicase YoaA and Its Interaction with Replication Clamp Loader Protein HolC in Escherichia coli.

The XP-D/DinG family of DNA helicases contributes to genomic stability in all three domains of life. Here, we investigate the role of one of these proteins, YoaA, of Escherichia coli. In E. coli, YoaA aids in tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor, and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolC's interaction with HolD and the rest of the replisome; YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions, we show that YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic, with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase-dependent induction by AZT is not blocked by lexA3 (Ind-), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3' nascent strand during replication; consistent with this, YoaA appears to aid in the removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. We provide evidence that YoaA and its paralog DinG may also initiate template switching that leads to deletions between tandem repeats in DNA. IMPORTANCE Maintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level. The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts.

In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study we isolate spontaneous suppressor mutants in a holCΔ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, and of sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (ß) and RpoC (ß') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.

The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming Replication/Transcription Conflicts.

In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp, and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability, although mutants show growth impairment, genetic instability, and sensitivity to DNA damaging agents. In this study, we isolate spontaneous suppressor mutants in a ΔholC strain and identify these by whole-genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, or alleles of sspA, encoding stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (ß), and RpoC (ß') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC, and sspA An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of Escherichia coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC's absence.

DNA damage-signaling, homologous recombination and genetic mutation induced by 5-azacytidine and DNA-protein crosslinks in Escherichia coli.

Covalent linkage between DNA and proteins produces highly toxic lesions and can be caused by commonly used chemotherapeutic agents, by internal and external chemicals and by radiation. In this study, using Escherichia coli, we investigate the consequences of 5-azacytidine (5-azaC), which traps covalent complexes between itself and the Dcm cytosine methyltransferase protein. DNA protein crosslink-dependent effects can be ascertained by effects that arise in wild-type but not in dcmΔ strains. We find that 5-azaC induces the bacterial DNA damage response and stimulates homologous recombination, a component of which is Dcm-dependent. Template-switching at an imperfect inverted repeat ("quasipalindrome", QP) is strongly enhanced by 5-azaC and this enhancement was entirely Dcm-dependent and independent of double-strand break repair. The SOS response helps ameliorate the mutagenic effect of 5-azaC but this is not a result of SOS-induced DNA polymerases since their induction, especially PolIV, seems to stimulate QP-associated mutagenesis. Cell division regulator SulA was also required for recovery of QP mutants induced by 5-azaC. In the absence of Lon protease, Dcm-dependent QP-mutagenesis is strongly elevated, suggesting it may play a role in DPC tolerance. Deletions at short tandem repeats, which occur likewise by a replication template-switch, are elevated, but only modestly, by 5-azaC. We see evidence for Dcm-dependent and-independent killing by 5-azaC in sensitive mutants, such as recA, recB, and lon; homologous recombination and deletion mutations are also stimulated in part by a Dcm-independent effect of 5-azaC. Whether this occurs by a different protein/DNA crosslink or by an alternative form of DNA damage is unknown.

Alternative complexes formed by the Escherichia coli clamp loader accessory protein HolC (x) with replication protein HolD (ψ) and repair protein YoaA.

Efficient and faithful replication of DNA is essential for all organisms. However, the replication fork frequently encounters barriers that need to be overcome to ensure cell survival and genetic stability. Cells must carefully balance and regulate replication vs. repair reactions. In Escherichia coli, the replisome consists of the DNA polymerase III holoenzyme, including DNA polymerase, proofreading exonuclease, processivity clamp and clamp loader, as well as a fork helicase, DnaB and primase, DnaG. We provide evidence here that one component of the clamp loader complex, HolC (or χ) plays a dual role via its ability to form 2 mutually exclusive complexes: one with HolD (or ψ) that recruits the clamp-loader and hence the DNA polymerase holoenzyme and another with helicase-like YoaA protein, a DNA-damage inducible repair protein. By yeast 2 hybrid analysis, we show that two residues of HolC, F64 and W57, at the interface in the structure with HolD, are required for interaction with HolD and for interaction with YoaA. Mutation of these residues does not interfere with HolC's interaction with single-strand DNA binding protein, SSB. In vivo, these mutations fail to complement the poor growth and sensitivity to azidothymidine, a chain-terminating replication inhibitor. In support of the notion that these are exclusive complexes, co-expression of HolC, HolD and YoaA, followed by pulldown of YoaA, yields a complex with HolC but not HolD. YoaA fails to pulldown HolC-F64A. We hypothesize that HolC, by binding with SSB, can recruit the DNA polymerase III holoenzyme through HolD, or an alternative repair complex with YoaA helicase.

Identifying Small Molecules That Promote Quasipalindrome-Associated Template-Switch Mutations in Escherichia coli.

DNA can assemble into non-B form structures that stall replication and cause genomic instability. One such secondary structure results from an inverted DNA repeat that can assemble into hairpin and cruciform structures during DNA replication. Quasipalindromes (QP), imperfect inverted repeats, are sites of mutational hotspots. Quasipalindrome-associated mutations (QPMs) occur through a template-switch mechanism in which the replicative polymerase stalls at a QP site and uses the nascent strand as a template instead of the correct template strand. This mutational event causes the QP to become a perfect or more perfect inverted repeat. Since it is not fully understood how template-switch events are stimulated or repressed, we designed a high-throughput screen to discover drugs that affect these events. QP reporters were engineered in the Escherichia coli lacZ gene to allow us to study template-switch events specifically. We tested 700 compounds from the NIH Clinical Collection through a disk diffusion assay and identified 11 positive hits. One of the hits was azidothymidine (zidovudine, AZT), a thymidine analog and DNA chain terminator. The other ten were found to be fluoroquinolone antibiotics, which induce DNA-protein crosslinks. This work shows that our screen is useful in identifying small molecules that affect quasipalindrome-associated template-switch mutations. We are currently assessing more small molecule libraries and applying this method to study other types of mutations.

Frequent template switching in postreplication gaps: suppression of deleterious consequences by the Escherichia coli Uup and RadD proteins.

When replication forks encounter template DNA lesions, the lesion is simply skipped in some cases. The resulting lesion-containing gap must be converted to duplex DNA to permit repair. Some gap filling occurs via template switching, a process that generates recombination-like branched DNA intermediates. The Escherichia coli Uup and RadD proteins function in different pathways to process the branched intermediates. Uup is a UvrA-like ABC family ATPase. RadD is a RecQ-like SF2 family ATPase. Loss of both functions uncovers frequent and RecA-independent deletion events in a plasmid-based assay. Elevated levels of crossing over and repeat expansions accompany these deletion events, indicating that many, if not most, of these events are associated with template switching in postreplication gaps as opposed to simple replication slippage. The deletion data underpin simulations indicating that multiple postreplication gaps may be generated per replication cycle. Both Uup and RadD bind to branched DNAs in vitro. RadD protein suppresses crossovers and Uup prevents nucleoid mis-segregation. Loss of Uup and RadD function increases sensitivity to ciprofloxacin. We present Uup and RadD as genomic guardians. These proteins govern two pathways for resolution of branched DNA intermediates such that potentially deleterious genome rearrangements arising from frequent template switching are averted.

Structure-Activity Relationship of Peptide-Conjugated Chloramphenicol for Inhibiting Escherichia coli.

Intravenous administration of a prodrug, chloramphenicol succinate (CLsu), is ineffective. Recently, we have shown that conjugation of diglycine of CLsu (CLsuGG) not only increases the antibiotic efficacy against Escherichia coli but also reduces adverse drug effects against bone marrow stromal cells. Here, we report the synthesis of structural analogues of CLsuGG and their activities against E. coli. These analogues reveal several trends: (i) except the water-insoluble analogues, the attachment of peptides to CLsu enhances the efficacy of the prodrugs; (ii) negative charges, high steric hindrance in the side chains, or a rigid diester decreases the activities of prodrugs in comparison to CLsuGG; (iii) dipeptides apparently increase the efficacy of the prodrugs most effectively; and so forth. This work suggests that conjugating peptides to CLsu effectively modulates the properties of prodrugs. The structure-activity relationship of these new conjugates may provide useful insights for expanding the pool of antibiotics.

Diglycine Enables Rapid Intrabacterial Hydrolysis for Activating Anbiotics against Gram-negative Bacteria.

Antimicrobial drug resistance demands novel approaches for improving the efficacy of antibiotics, especially against Gram-negative bacteria. Herein, we report that conjugating a diglycine (GG) to an antibiotic prodrug drastically accelerates intrabacterial ester-bond hydrolysis required for activating the antibiotic. Specifically, the attachment of GG to chloramphenicol succinate (CLsu) generates CLsuGG, which exhibits about an order of magnitude higher inhibitory efficacy than CLsu against Escherichia coli. Further studies reveal that CLsuGG undergoes rapid hydrolysis, catalyzed by intrabacterial esterases (e.g., BioH and YjfP), to generate chloramphenicol (CL) in E. coli. Importantly, the conjugate exhibits lower cytotoxicity to bone marrow stromal cells than CL. Structural analogues of CLsuGG indicate that the conjugation of GG to an antibiotic prodrug is an effective strategy for accelerating enzymatic prodrug hydrolysis and enhancing the antibacterial efficacy of antibiotics.

Stimulation of Replication Template-Switching by DNA-Protein Crosslinks.

Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-Azacytidine (5-azaC) is a potent mutagen for template-switching. This effect is dependent on DNA cytosine methylase (Dcm), implicating the Dcm-DNA covalent complex trapped by 5-azaC as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, which can be explained by blocks to the replicative helicase and/or fork regression. We find that template-switch mutagenesis induced by 5-azaC does not require double strand break repair via RecABCD; the ability to induce the SOS response is anti-mutagenic. Mutants in recB, but not recA, exhibit high constitutive rates of template-switching, and we suggest that RecBCD-mediated DNA degradation prevents template-switching associated with fork regression. A mutation in the DnaB fork helicase also promotes high levels of template-switching. We also find that other DPC-inducers, formaldehyde (a non-specific crosslinker) and ciprofloxacin (a topoisomerase II poison) are also strong mutagens for template-switching with similar genetic properties. Induction of mutations and genetic rearrangements that occur by template-switching may constitute a previously unrecognized component of the genotoxicity and genetic instability promoted by DPCs.

Between sisters: Watching replication-associated recombinational DNA repair.

Amarh et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201803020) visualize for the first time the repair of double-strand breaks during DNA replication. As viewed by live-cell fluorescent imaging of Escherichia coli, repair of replication-dependent breaks is extraordinarily rapid and localized within the cell.

SSB recruitment of Exonuclease I aborts template-switching in Escherichia coli.

Misalignment of a nascent strand and the use of an alternative template during DNA replication, a process termed "template-switching", can give rise to frequent mutations and genetic rearrangements. Mutational hotspots are frequently found associated with imperfect inverted repeats ("quasipalindromes" or "QPs") in many organisms, including bacteriophage, bacteria, yeast and mammals. Evidence suggests that QPs mutate by a replication template-switch whereby one copy of the inverted repeat templates synthesis of the other. To study quasipalindrome-associated mutagenesis ("QPM") more systematically, we have engineered mutational reporters in the lacZ gene of Escherichia coli, that revert to Lac+ specifically by QPM. We and others have shown that QPM is more efficient during replication of the leading strand than it is on the lagging strand. We have previously shown that QPM is elevated and that the leading-strand bias is lost in mutants lacking the major 3' ssDNA exonucleases, ExoI and ExoVII. This suggests that one or both of these exonucleases more efficiently abort template-switches on the lagging strand. Here, we show that ExoI is primarily responsible for this bias and that its ability to be recruited by single-strand DNA binding protein plays a critical role in QPM avoidance and strand bias. In addition to these stand-alone exonucleases, loss of the 3' proofreading exonuclease activity of the replicative DNA polymerase III also greatly elevates QPM. This may be because template-switching is initiated by base misincorporation, leading to polymerase dissociation and subsequent nascent strand misalignment; alternatively or additionally, the proofreading exonuclease may scavenge displaced 3' DNA that would otherwise be free to misalign.

Template-switching during replication fork repair in bacteria.

Replication forks frequently are challenged by lesions on the DNA template, replication-impeding DNA secondary structures, tightly bound proteins or nucleotide pool imbalance. Studies in bacteria have suggested that under these circumstances the fork may leave behind single-strand DNA gaps that are subsequently filled by homologous recombination, translesion DNA synthesis or template-switching repair synthesis. This review focuses on the template-switching pathways and how the mechanisms of these processes have been deduced from biochemical and genetic studies. I discuss how template-switching can contribute significantly to genetic instability, including mutational hotspots and frequent genetic rearrangements, and how template-switching may be elicited by replication fork damage.

Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli.

RadA (also known as 'Sms') is a highly conserved protein, found in almost all eubacteria and plants, with sequence similarity to the RecA strand exchange protein and a role in homologous recombination. We investigate here the biochemical properties of the E. coli RadA protein and several mutant forms. RadA is a DNA-dependent ATPase, a DNA-binding protein and can stimulate the branch migration phase of RecA-mediated strand transfer reactions. RadA cannot mediate synaptic pairing between homologous DNA molecules but can drive branch migration to extend the region of heteroduplex DNA, even without RecA. Unlike other branch migration factors RecG and RuvAB, RadA stimulates branch migration within the context of the RecA filament, in the direction of RecA-mediated strand exchange. We propose that RadA-mediated branch migration aids recombination by allowing the 3' invading strand to be incorporated into heteroduplex DNA and to be extended by DNA polymerases.

Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein.

Elongating DNA polymerases frequently encounter lesions or structures that impede progress and require repair before DNA replication can be completed. Therefore, directing repair factors to a blocked fork, without interfering with normal replication, is important for proper cell function, and it is a process that is not well understood. To study this process, we have employed the chain-terminating nucleoside analog, 3' azidothymidine (AZT) and the E. coli genetic system, for which replication and repair factors have been well-defined. By using high-expression suppressor screens, we identified yoaA, encoding a putative helicase, and holC, encoding the Chi component of the replication clamp loader, as genes that promoted tolerance to AZT. YoaA is a putative Fe-S helicase in the XPD/RAD3 family for which orthologs can be found in most bacterial genomes; E. coli has a paralog to YoaA, DinG, which possesses 5' to 3' helicase activity and an Fe-S cluster essential to its activity. Mutants in yoaA are sensitive to AZT exposure; dinG mutations cause mild sensitivity to AZT and exacerbate the sensitivity of yoaA mutant strains. Suppression of AZT sensitivity by holC or yoaA was mutually codependent and we provide evidence here that YoaA and Chi physically interact. Interactions of Chi with single-strand DNA binding protein (SSB) and with Psi were required to aid AZT tolerance, as was the proofreading 3' exonuclease, DnaQ. Our studies suggest that repair is coupled to blocked replication through these interactions. We hypothesize that SSB, through Chi, recruits the YoaA helicase to replication gaps and that unwinding of the nascent strand promotes repair and AZT excision. This recruitment prevents the toxicity of helicase activity and aids the handoff of repair with replication factors, ensuring timely repair and resumption of replication.

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.