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.

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.

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.

Ethylene oxide and propylene oxide derived N7-alkylguanine adducts are bypassed accurately in vivo.

Adducts formed at the nucleophilic N7 position of guanine are the most abundant lesions produced by alkylating agents such as ethylene oxide (EO) and propylene oxide (PO). In order to investigate the intrinsic mutagenic potential of N7-alkylguanine adducts, we prepared single-stranded DNA probes containing a single well-defined N7-alkylguanine adduct under conditions that minimize the presence of depurinated molecules. Following introduction of these probes into Escherichia coli cells, the effect of the N7-alkylguanine adducts on the efficiency and fidelity of replication was determined. To investigate the effect on replication we monitored the relative transformation efficiency of the lesion containing constructs with respect to the control construct. The methyl adduct was found not to be toxic, while the N7-(2-hydroxyethyl)guanine (N7-heG) and N7-(2-hydroxypropyl)guanine (N7-hpG) adducts reduce the transformation efficiency to ≈70% and 40%, respectively. Within the detection limits of our assay, replication across the N7-alkylguanine adducts in vivo is essentially error-free, as no mutant colony was observed among ≈300 individual sequenced colonies (i.e., mutation frequency<0.3%).

DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts.

Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N(2)-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same ß clamp and to positively dissociate Pol III from ß clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (-)-trans-anti-benzo[a]pyrene-N(2)-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.

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.

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O6-alkylguanine adducts in Escherichia coli.

O(6)-alkylG adducts are highly mutagenic due to their capacity to efficiently form O(6)-alkylG:T mispairs during replication, thus triggering G→A transitions. Mutagenesis is largely prevented by repair strategies such as reversal by alkyltransferases or excision by nucleotide excision repair (NER). Moreover, methyl-directed mismatch repair (MMR) is known to trigger sensitivity to methylating agents via a mechanism that involves recognition by MutS of the O(6)-mG:T replication intermediates. We wanted to investigate the mechanism by which MMR controls the genotoxicity of environmentally relevant O(6)-alkylG adducts formed by ethylene oxide and propylene oxide. Recently, the alkyltransferase-like gene ybaZ (eATL) was shown to enhance repair of these slightly larger O(6)-alkylG adducts by NER. We analyzed the toxicity and mutagenesis induced by these O(6)-alkylG adducts using single-adducted plasmid probes. We show that the eATL gene product prevents MMR-mediated attack of the O(6)-alkylG:T replication intermediate for the larger alkyl groups but not for methyl. In vivo data are compatible with the occurrence of repeated cycles of MMR attack of the O(6)-alkylG:T intermediate. In addition, in vitro, the eATL protein efficiently prevents binding of MutS to the O(6)-alkylG:T mispairs formed by the larger alkyl groups but not by methyl. In conclusion, eATL not only enhances the efficiency of repair of these larger adducts by NER, it also shields these adducts from MMR-mediated toxicity.

Rad8Rad5/Mms2-Ubc13 ubiquitin ligase complex controls translesion synthesis in fission yeast.

Many DNA lesions cause pausing of replication forks at lesion sites; thus, generating gaps in the daughter strands that are filled-in by post-replication repair (PRR) pathways. In Saccharomyces cerevisiae, PRR involves translesion synthesis (TLS) mediated by Poleta or Polzeta, or Rad5-dependent gap filling through a poorly characterized error-free mechanism. We have developed an assay to monitor error-free and mutagenic TLS across single DNA lesions in Schizosaccharomyces pombe. For both main UV photolesions, we have delineated a major error-free pathway mediated by a distinct combination of TLS polymerases. Surprisingly, these TLS pathways require enzymes needed for poly-ubiquitination of proliferating cell nuclear antigen (PCNA) as well as those required for mono-ubiquitination. For pathways that require several TLS polymerases the poly-ubiquitin chains of PCNA may facilitate their recruitment through specific interactions with their multiple ubiquitin-binding motifs. These error-free TLS pathways may at least partially account for the previously described poly-ubiquitination-dependent error-free branch of PRR. This work highlights major differences in the control of lesion tolerance pathways between S. pombe and S. cerevisiae despite the homologous sets of PRR genes these organisms share.

The alkyltransferase-like ybaZ gene product enhances nucleotide excision repair of O(6)-alkylguanine adducts in E. coli.

O(6)-methylguanine adducts are potent pre-mutagenic lesions owing to their high capacity to direct mis-insertion of thymine when bypassed by replicative DNA polymerases. The strong mutagenic potential of these adducts is prevented by alkyltransferases such as Ada and Ogt in Escherichia coli that transfer the methyl group to one of their cysteine residues. Alkyl residues larger than methyl are generally weak substrates for reversion by alkyltransferases. In this paper we have investigated the genotoxic potential of the O(6)-alkylguanine adducts formed by ethylene and propylene oxide using single-adducted plasmid probes. Our work shows that the ybaZ gene product, a member of the alkyltransferase-like protein family, strongly enhances the repair by nucleotide excision repair of the larger O(6)-alkylguanine adducts that are otherwise poor substrates for alkyltransferases. The YbaZ protein is shown to interact with UvrA. This factor may thus enhance the efficiency of nucleotide excision repair in a way similar to the Transcription-Repair Coupling factor Mfd, by recruiting the UvrA(2).UvrB complex to the adduct site via its interaction with UvrA.