Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.

Structure-specific endonucleases resolve DNA secondary structures generated during DNA repair and recombination. The yeast 5' flap endonuclease Slx1-Slx4 has received particular attention with the finding that Slx4 has Slx1-independent key functions in genome maintenance. Although Slx1 is a highly conserved protein in eukaryotes, no orthologs of Slx4 were reported other than in fungi. Here we report the identification of Slx4 orthologs in metazoa, including fly MUS312, essential for meiotic recombination, and human BTBD12, an ATM/ATR checkpoint kinase substrate. Human SLX1-SLX4 displays robust Holliday junction resolvase activity in addition to 5' flap endonuclease activity. Depletion of SLX1 and SLX4 results in 53BP1 foci accumulation and H2AX phosphorylation as well as cellular hypersensitivity to MMS. Furthermore, we show that SLX4 binds the XPF(ERCC4) and MUS81 subunits of the XPF-ERCC1 and MUS81-EME1 endonucleases and is required for DNA interstrand crosslink repair. We propose that SLX4 acts as a docking platform for multiple structure-specific endonucleases.

Accidental Encounter of Repair Intermediates in Alkylated DNA May Lead to Double-Strand Breaks in Resting Cells.

In clinics, chemotherapy is often combined with surgery and radiation to increase the chances of curing cancers. In the case of glioblastoma (GBM), patients are treated with a combination of radiotherapy and TMZ over several weeks. Despite its common use, the mechanism of action of the alkylating agent TMZ has not been well understood when it comes to its cytotoxic effects in tumor cells that are mostly non-dividing. The cellular response to alkylating DNA damage is operated by an intricate protein network involving multiple DNA repair pathways and numerous checkpoint proteins that are dependent on the type of DNA lesion, the cell type, and the cellular proliferation state. Among the various alkylating damages, researchers have placed a special on O6-methylguanine (O6-mG). Indeed, this lesion is efficiently removed via direct reversal by O6-methylguanine-DNA methyltransferase (MGMT). As the level of MGMT expression was found to be directly correlated with TMZ efficiency, O6-mG was identified as the critical lesion for TMZ mode of action. Initially, the mode of action of TMZ was proposed as follows: when left on the genome, O6-mG lesions form O6-mG: T mispairs during replication as T is preferentially mis-inserted across O6-mG. These O6-mG: T mispairs are recognized and tentatively repaired by a post-replicative mismatched DNA correction system (i.e., the MMR system). There are two models (futile cycle and direct signaling models) to account for the cytotoxic effects of the O6-mG lesions, both depending upon the functional MMR system in replicating cells. Alternatively, to explain the cytotoxic effects of alkylating agents in non-replicating cells, we have proposed a "repair accident model" whose molecular mechanism is dependent upon crosstalk between the MMR and the base excision repair (BER) systems. The accidental encounter between these two repair systems will cause the formation of cytotoxic DNA double-strand breaks (DSBs). In this review, we summarize these non-exclusive models to explain the cytotoxic effects of alkylating agents and discuss potential strategies to improve the clinical use of alkylating agents.

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes.

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80-100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

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.

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.

FF483-484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance.

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483-484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.

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.

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.

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.

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.

Increase in dNTP pool size during the DNA damage response plays a key role in spontaneous and induced-mutagenesis in Escherichia coli.

Exposure of Escherichia coli to UV light increases expression of NrdAB, the major ribonucleotide reductase leading to a moderate increase in dNTP levels. The role of elevated dNTP levels during translesion synthesis (TLS) across specific replication-blocking lesions was investigated. Here we show that although the specialized DNA polymerase PolV is necessary for replication across UV-lesions, such as cyclobutane pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproduct, Pol V per se is not sufficient. Indeed, efficient TLS additionally requires elevated dNTP levels. Similarly, for the bypass of an N-2-acetylaminofluorene-guanine adduct that requires Pol II instead of PolV, efficient TLS is only observed under conditions of high dNTP levels. We suggest that increased dNTP levels transiently modify the activity balance of Pol III (i.e., increasing the polymerase and reducing the proofreading functions). Indeed, we show that the stimulation of TLS by elevated dNTP levels can be mimicked by genetic inactivation of the proofreading function (mutD5 allele). We also show that spontaneous mutagenesis increases proportionally to dNTP pool levels, thus defining a unique spontaneous mutator phenotype. The so-called "dNTP mutator" phenotype does not depend upon any of the specialized DNA polymerases, and is thus likely to reflect an increase in Pol III's own replication errors because of the modified activity balance of Pol III. As up-regulation of the dNTP pool size represents a common physiological response to DNA damage, the present model is likely to represent a general and unique paradigm for TLS pathways in many organisms.

Escherichia coli DNA polymerase III is responsible for the high level of spontaneous mutations in mutT strains.

Reactive oxygen species induce oxidative damage in DNA precursors, i.e. dNTPs, leading to point mutations upon incorporation. Escherichia coli mutT strains, deficient in the activity hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), display more than a 100-fold higher spontaneous mutation frequency over the wild-type strain. 8-oxo-dGTP induces A to C transversions when misincorporated opposite template A. Here, we report that DNA pol III incorporates 8-oxo-dGTP ≈ 20 times more efficiently opposite template A compared with template C. Single, double or triple deletions of pol I, pol II, pol IV or pol V had modest effects on the mutT mutator phenotype. Only the deletion of all four polymerases led to a 70% reduction of the mutator phenotype. While pol III may account for nearly all 8-oxo-dGTP incorporation opposite template A, it only extends ≈ 30% of them, the remaining 70% being extended by the combined action of pol I, pol II, pol IV or pol V. The unique property of pol III, a C-family DNA polymerase present only in eubacteria, to preferentially incorporate 8-oxo-dGTP opposite template A during replication might explain the high spontaneous mutation frequency in E. coli mutT compared with the mammalian counterparts lacking the 8-oxo-dGTP hydrolysing activities.

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.

Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts.

In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.

Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation.

In Escherichia coli, it is genetically well established that the beta-clamp and RecA are essential cofactors that endow DNA polymerase (Pol) V with lesion bypass activity. However, the biochemical basis for these requirements is still largely unknown. Because the process of translesion synthesis (TLS) requires that the specialized DNA polymerase synthesize in a single binding event a TLS patch that is long enough to resist external proofreading, it is critical to monitor Pol V burst synthesis. Here, we dissect the distinct roles that RecA and the beta-clamp perform during the Pol V activation process using physiologically relevant long single-stranded template DNA, similar to those used in genetic assays. Our data show that the beta-clamp endows the complex between Pol V and the template DNA with increased stability. Also, the RecA filament formed in cis on the single-stranded DNA produced downstream from the lesion stretches the template DNA to allow smooth elongation of the nascent strand by Pol V. The concurrent action of both cofactors is required for achieving productive TLS events. The present article presents an integrated view of TLS under physiologically relevant conditions in E. coli that may represent a paradigm for lesion bypass in other organisms.

Double-strand breaks: When DNA repair events accidentally meet.

The cellular response to alkylation damage is complex, involving multiple DNA repair pathways and checkpoint proteins, depending on the DNA lesion, the cell type, and the cellular proliferation state. The repair of and response to O-alkylation damage, primarily O6-methylguaine DNA adducts (O6-mG), is the purview of O6-methylguanine-DNA methyltransferase (MGMT). Alternatively, this lesion, if left un-repaired, induces replication-dependent formation of the O6-mG:T mis-pair and recognition of this mis-pair by the post-replication mismatch DNA repair pathway (MMR). Two models have been suggested to account for MMR and O6-mG DNA lesion dependent formation of DNA double-strand breaks (DSBs) and the resulting cytotoxicity - futile cycling and direct DNA damage signaling. While there have been hints at crosstalk between the MMR and base excision repair (BER) pathways, clear mechanistic evidence for such pathway coordination in the formation of DSBs has remained elusive. However, using a novel protein capture approach, Fuchs and colleagues have demonstrated that DSBs result from an encounter between MMR-induced gaps initiated at alkylation induced O6-mG:C sites and BER-induced nicks at nearby N-alkylation adducts in the opposite strand. The accidental encounter between these two repair events is causal in the formation of DSBs and the resulting cellular response, documenting a third model to account for O6-mG induced cell death in non-replicating cells. This graphical review highlights the details of this Repair Accident model, as compared to current models, and we discuss potential strategies to improve clinical use of alkylating agents such as temozolomide, that can be inferred from the Repair Accident model.

Enrichment of plasmid pool from E. coli for mass sequencing to detect untargeted mutagenic events.

Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a source of genetic diversity. Identifying such untargeted mutations in vivo is challenging due to the bulk of DNA that does not undergo TLS. Here, we present a protocol to enrich a plasmid pool that underwent Pol V-mediated TLS in Escherichia coli for mass sequencing. The concept of this protocol could be applied into any species. For complete details on the use and execution of this protocol, please refer to Isogawa et al. (2018).

Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide.

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.

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.