Rad51 determines pathway usage in post-replication repair.

Stalled replication forks can be processed by several distinct mechanisms collectively called post-replication repair which includes homologous recombination, fork regression, and translesion DNA synthesis. However, the regulation of the usage between these pathways is not fully understood. The Rad51 protein plays a pivotal role in maintaining genomic stability through its roles in HR and in protecting stalled replication forks from degradation. We report the isolation of separation-of-function mutations in Saccharomyces cerevisiae Rad51 that retain their recombination function but display a defect in fork protection leading to a shift in post-replication repair pathway usage from HR to alternate pathways including mutagenic translesion synthesis. Rad51-E135D and Rad51-K305N show normal in vivo and in vitro recombination despite changes in their DNA binding profiles, in particular to dsDNA, with a resulting effect on their ATPase activities. The mutants lead to a defect in Rad51 recruitment to stalled forks in vivo as well as a defect in the protection of dsDNA from degradation by Dna2-Sgs1 and Exo1 in vitro . A high-resolution cryo-electron microscopy structure of the Rad51-ssDNA filament at 2.4 Å resolution provides a structural basis for a mechanistic understanding of the mutant phenotypes. Together, the evidence suggests a model in which Rad51 binding to duplex DNA is critical to control pathway usage at stalled replication forks.

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS.

Processivity clamps mediate polymerase switching for translesion synthesis (TLS). All three E. coli TLS polymerases interact with the ß2 processivity clamp through a conserved clamp-binding motif (CBM), which is indispensable for TLS. Notably, Pol IV also makes a unique secondary contact with the clamp through non-CBM residues. However, the role of this "rim contact" in Pol IV-mediated TLS remains poorly understood. Here we show that the rim contact is critical for TLS past strong replication blocks. In in vitro reconstituted Pol IV-mediated TLS, ablating the rim contact compromises TLS past 3-methyl dA, a strong block, while barely affecting TLS past N2-furfuryl dG, a weak block. Similar observations are also made in E. coli cells bearing a single copy of these lesions in the genome. Within lesion-stalled replication forks, the rim interaction and ssDNA binding protein cooperatively poise Pol IV to better compete with Pol III for binding to a cleft through its CBM. We propose that this bipartite clamp interaction enables Pol IV to rapidly resolve lesion-stalled replication through TLS at the fork, which reduces damage induced mutagenesis.

Rad5 participates in lesion bypass through its Rev1-binding and ubiquitin ligase domains, but not through its helicase function.

DNA Damage Tolerance (DDT) functions to bypass replication-blocking lesions and is divided into two distinct pathways: error-prone Translesion Synthesis (TLS) and error-free Damage Avoidance (DA). Rad5 is a multifunctional protein that is involved in these DDT processes. Saccharomyces cerevisiae Rad5 contains three well defined domains: a RING domain that promotes PCNA polyubiquitination, a ssDNA-dependent ATPase/helicase domain, and a Rev1-binding domain. Both the RING domain and the ATPase/helicase domain are conserved in human Rad5 ortholog HLTF. In this study we used domain-specific mutants to address the contribution of each of the Rad5 domains to the lesion tolerance. We demonstrate that the two critical functions of Rad5 during DNA damage tolerance are the activation of template switching through polyubiquitination of PCNA and the recruitment of TLS polymerases, and that loss of one of those functions can be compensated by increased usage of the other. We also show that, unlike previously suggested, the helicase activity does not play any role in lesion tolerance.

Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis.

Processivity clamps tether DNA polymerases to DNA, allowing their access to the primer-template junction. In addition to DNA replication, DNA polymerases also participate in various genome maintenance activities, including translesion synthesis (TLS). However, owing to the error-prone nature of TLS polymerases, their association with clamps must be tightly regulated. Here we show that fork-associated ssDNA-binding protein (SSB) selectively enriches the bacterial TLS polymerase Pol IV at stalled replication forks. This enrichment enables Pol IV to associate with the processivity clamp and is required for TLS on both the leading and lagging strands. In contrast, clamp-interacting proteins (CLIPs) lacking SSB binding are spatially segregated from the replication fork, minimally interfering with Pol IV-mediated TLS. We propose that stalling-dependent structural changes within clusters of fork-associated SSB establish hierarchical access to the processivity clamp. This mechanism prioritizes a subset of CLIPs with SSB-binding activity and facilitates their exchange at the replication fork.

Single strand gap repair: The presynaptic phase plays a pivotal role in modulating lesion tolerance pathways.

During replication, the presence of unrepaired lesions results in the formation of single stranded DNA (ssDNA) gaps that need to be repaired to preserve genome integrity and cell survival. All organisms have evolved two major lesion tolerance pathways to continue replication: Translesion Synthesis (TLS), potentially mutagenic, and Homology Directed Gap Repair (HDGR), that relies on homologous recombination. In Escherichia coli, the RecF pathway repairs such ssDNA gaps by processing them to produce a recombinogenic RecA nucleofilament during the presynaptic phase. In this study, we show that the presynaptic phase is crucial for modulating lesion tolerance pathways since the competition between TLS and HDGR occurs at this stage. Impairing either the extension of the ssDNA gap (mediated by the nuclease RecJ and the helicase RecQ) or the loading of RecA (mediated by RecFOR) leads to a decrease in HDGR and a concomitant increase in TLS. Hence, we conclude that defects in the presynaptic phase delay the formation of the D-loop and increase the time window allowed for TLS. In contrast, we show that a defect in the postsynaptic phase that impairs HDGR does not lead to an increase in TLS. Unexpectedly, we also reveal a strong genetic interaction between recF and recJ genes, that results in a recA deficient-like phenotype in which HDGR is almost completely abolished.

Eukaryotic stress-induced mutagenesis is limited by a local control of translesion synthesis.

The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this work, we raise the question whether TLS is controlled locally or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that (i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, (ii) a single lesion is enough to induce PCNA ubiquitination and (iii) PCNA ubiquitination is imperative for TLS to occur. More importantly, we show that the activation of the DDR that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.

iDamage: a method to integrate modified DNA into the yeast genome.

In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of Saccharomyces cerevisiae, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of Rad51 recombinase) or through its control by preventing the polyubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of Trans Lesion Synthesis (TLS). Such regulatory aspects of DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed DNA lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will enable the exploration of many biological processes related to replication and repair of modified DNA.

DNA lesions proximity modulates damage tolerance pathways in Escherichia coli.

The genome of all organisms is constantly threatened by numerous agents that cause DNA damage. When the replication fork encounters an unrepaired DNA lesion, two DNA damage tolerance pathways are possible: error-prone translesion synthesis (TLS) that requires specialized DNA polymerases, and error-free damage avoidance that relies on homologous recombination (HR). The balance between these two mechanisms is essential since it defines the level of mutagenesis during lesion bypass, allowing genetic variability and adaptation to the environment, but also introduces the risk of generating genome instability. Here we report that the mere proximity of replication-blocking lesions that arise in Escherichia coli's genome during a genotoxic stress leads to a strong increase in the use of the error-prone TLS. We show that this increase is caused by the local inhibition of HR due to the overlapping of single-stranded DNA regions generated downstream of the lesions. This increase in TLS is independent of SOS activation, but its mutagenic effect is additive with the one of SOS. Hence, the combination of SOS induction and lesions proximity leads to a strong increase in TLS that becomes the main lesion tolerance pathway used by the cell during a genotoxic stress.

Inserting Site-Specific DNA Lesions into Whole Genomes.

Here, we describe a methodology that allows the insertion of site-specific DNA lesions into genomes in living cells. The technique involves the integration of a plasmid containing a site-specific lesion engineered in vitro into a precise location in the genome via the site-specific recombination reaction from phage lambda. The notion of DNA lesion is not restricted to chemically modified nucleotides but also refers to unusual DNA structures. This method will be instrumental to study qualitatively and quantitatively the genetic consequences of site-specific lesions in vivo; moreover, it does also allow analyzing the molecular structure of stalled replication forks at well-defined locations.

A non-catalytic role of RecBCD in homology directed gap repair and translesion synthesis.

The RecBCD complex is a key factor in DNA metabolism. This protein complex harbors a processive nuclease and two helicases activities that give it the ability to process duplex DNA ends. These enzymatic activities make RecBCD a major player in double strand break repair, conjugational recombination and degradation of linear DNA. In this work, we unravel a new role of the RecBCD complex in the processing of DNA single-strand gaps that are generated at DNA replication-blocking lesions. We show that independently of its nuclease or helicase activities, the entire RecBCD complex is required for recombinational repair of the gap and efficient translesion synthesis. Since none of the catalytic functions of RecBCD are required for those processes, we surmise that the complex acts as a structural element that stabilizes the blocked replication fork, allowing efficient DNA damage tolerance.

A defect in homologous recombination leads to increased translesion synthesis in E. coli.

DNA damage tolerance pathways allow cells to duplicate their genomes despite the presence of replication blocking lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS) and homology directed gap repair (HDGR). TLS pathways involve specialized DNA polymerases that are able to synthesize past DNA lesions with an intrinsic risk of causing point mutations. In contrast, HDGR pathways are essentially error-free as they rely on the recovery of missing information from the sister chromatid by RecA-mediated homologous recombination. We have investigated the genetic control of pathway choice between TLS and HDGR in vivo in Escherichia coli In a strain with wild type RecA activity, the extent of TLS across replication blocking lesions is generally low while HDGR is used extensively. Interestingly, recA alleles that are partially impaired in D-loop formation confer a decrease in HDGR and a concomitant increase in TLS. Thus, partial defect of RecA's capacity to invade the homologous sister chromatid increases the lifetime of the ssDNA.RecA filament, i.e. the 'SOS signal'. This increase favors TLS by increasing both the TLS polymerase concentration and the lifetime of the TLS substrate, before it becomes sequestered by homologous recombination. In conclusion, the pathway choice between error-prone TLS and error-free HDGR is controlled by the efficiency of homologous recombination.

Single-strand gap repair involves both RecF and RecBCD pathways.

Homologous recombination repairs discontinuities in DNA including single-strand gaps (SSGs) and double-strand breaks (DSBs). This commentary describes how the RecBCD and RecF pathways might be exchangeable for the repair of their respective DSB and SSG canonical substrates. In particular, I will discuss how the RecBCD pathway could engage in the repair of an SSG even when the latter is not associated with a DSB.

Bacterial Proliferation: Keep Dividing and Don't Mind the Gap.

DNA Damage Tolerance (DDT) mechanisms help dealing with unrepaired DNA lesions that block replication and challenge genome integrity. Previous in vitro studies showed that the bacterial replicase is able to re-prime downstream of a DNA lesion, leaving behind a single-stranded DNA gap. The question remains of what happens to this gap in vivo. Following the insertion of a single lesion in the chromosome of a living cell, we showed that this gap is mostly filled in by Homology Directed Gap Repair in a RecA dependent manner. When cells fail to repair this gap, or when homologous recombination is impaired, cells are still able to divide, leading to the loss of the damaged chromatid, suggesting that bacteria lack a stringent cell division checkpoint mechanism. Hence, at the expense of losing one chromatid, cell survival and proliferation are ensured.

Chronology in lesion tolerance gives priority to genetic variability.

The encounter of a replication fork with a blocking DNA lesion is a common event that cells need to address properly to preserve genome integrity. Cells possess two main strategies to tolerate unrepaired lesions: potentially mutagenic translesion synthesis (TLS) and nonmutagenic damage avoidance (DA). Little is known about the partitioning between these two strategies. Because genes involved in DA mechanisms (i.e., recA) are expressed early and genes involved in TLS (i.e., Pol V) are expressed late during the bacterial SOS response, it has long been thought that TLS was the last recourse to bypass DNA lesions when repair and nonmutagenic DA mechanisms have failed. By using a recently described methodology, we followed the fate of a single replication-blocking lesion introduced in the Escherichia coli genome during acute genotoxic stress. We show that lesion tolerance events (i) only occur when the SOS response is fully induced and (ii) are executed in chronological order, with TLS coming first, followed by DA. Therefore, in response to genotoxic stress, bacterial cells give priority to TLS, a minor pathway able to generate genetic diversity before implementing the major nonmutagenic pathway that ensures survival.

Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome.

Although most deoxyribonucleic acid (DNA) lesions are accurately repaired before replication, replication across unrepaired lesions is the main source of point mutations. The lesion tolerance processes, which allow damaged DNA to be replicated, entail two branches, error-prone translesion synthesis (TLS) and error-free damage avoidance (DA). While TLS pathways are reasonably well established, DA pathways are poorly understood. The fate of a replication-blocking lesion is generally explored by means of plasmid-based assays. Although such assays represent efficient tools to analyse TLS, we show here that plasmid-borne lesions are inappropriate models to study DA pathways due to extensive replication fork uncoupling. This observation prompted us to develop a method to graft, site-specifically, a single lesion in the genome of a living cell. With this novel assay, we show that in Escherichia coli DA events massively outweigh TLS events and that in contrast to plasmid, chromosome-borne lesions partially require RecA for tolerance.

Yeast Rev1 protein promotes complex formation of DNA polymerase zeta with Pol32 subunit of DNA polymerase delta.

Yeast DNA polymerase (Pol) delta, essential for DNA replication, is comprised of 3 subunits, Pol3, Pol31, and Pol32. Of these, the catalytic subunit Pol3 and the second subunit Pol31 are essential, whereas the Pol32 subunit is not essential for DNA replication. Although Pol32 is an integral component of Pol delta, it is also required for translesion synthesis (TLS) by Pol zeta. To begin to decipher the bases of Pol32 involvement in Pol zeta-mediated TLS, here we examine whether Pol32 physically interacts with Pol zeta or its associated proteins and provide evidence for the physical interaction of Pol32 with Rev1. Rev1 plays an indispensable structural role in Pol zeta-mediated TLS and it binds the Rev3 catalytic subunit of Pol zeta. Here, we show that although Pol32 does not directly bind Pol zeta, Pol32 can bind the Rev1-Pol zeta complex through its interaction with Rev1. We find that Pol32 binding has no stimulatory effect on DNA synthesis either by Rev1 in the Rev1-Pol32 complex or by Pol zeta in the Pol zeta-Rev1-Pol32 complex, irrespective of whether proliferating cell nuclear antigen has been loaded onto DNA or not. We discuss evidence for the biological significance of Rev1 binding to Pol32 for Pol zeta function in TLS and suggest a structural role for Rev1 in modulating the binding of Pol zeta with Pol32 in Pol delta stalled at a lesion site.

Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast.

Unrepaired DNA lesions in the template strand block the replication fork. In yeast, Mec1 protein kinase-mediated replication checkpoint prevents the breakdown of replication forks and maintains viability in DNA-damaged cells going through the S phase. By ensuring that the replisome does not dissociate from the fork stalled at the lesion site, the replication checkpoint presumably coordinates the action of lesion bypass processes with the replisome. However, it has remained unclear as to which of the lesion bypass processes-translesion synthesis (TLS) and/or template switching-depend on the activation of the replication checkpoint. Here we determine if the Mec1 kinase and the subunits of the checkpoint clamp and the clamp loader are required for TLS. We show that proficient TLS can occur in the absence of these checkpoint proteins in nucleotide excision repair (NER)-proficient cells; however, in the absence of NER, checkpoint protein-mediated Rev1 phosphorylation contributes to increasing the proficiency of DNA polymerase zeta-dependent TLS.

Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae.

In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases eta or zeta or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities--a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polzeta. Using duplex plasmids carrying different site-specific DNA lesions--an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct--we show that Rad5 is needed for Polzeta-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.

Mutational specificity and genetic control of replicative bypass of an abasic site in yeast.

Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA polymerases (Pols). Here we determine the mutational specificity and the genetic control of translesion synthesis (TLS) opposite an AP site in yeast by using a double-stranded plasmid system that we have devised in which bidirectional replication proceeds from a replication origin. We find that the rate, the genetic control, and the types and frequencies of nucleotides inserted opposite the AP site are very similar for both the leading and the lagging DNA strands, and that an A is predominantly inserted opposite the AP site, whereas C insertion by Rev1 constitutes a much less frequent event. In striking contrast, in studies that have been reported previously for AP bypass with gapped-duplex and single-stranded plasmids, it has been shown that a C is the predominant nucleotide inserted opposite the AP site. We discuss the implications of our observations for the mechanisms of TLS on the leading versus the lagging DNA strand and suggest that lesion bypass during replication involves the coordination of activities of the replicative Pol with that of the lesion-bypass Pol.