Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.     

A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family

The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5′ phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.     

SNM1B/Apollo interacts with Astrin and is required for the prophase cell cycle checkpoint

Previously, we have shown that SNM1A is a multifunctional gene involved in both the DNA damage response and in an early mitotic checkpoint in response to spindle stress. Another member of the SNM1 gene family, SNM1B/Apollo, has been shown to have roles in both the response to DNA interstrand cross-linking agents and in telomere protection during S phase. Here, we demonstrate a novel role for SNM1B/Apollo in mitosis in response to spindle stress. SNM1B-deficient cells exhibit a defect in the prophase checkpoint. Loss of the prophase checkpoint induces an extended mitotic delay, which is due to prolonged activation of the spindle checkpoint. In addition, we show that SNM1B/Apollo interacts with the essential microtubule binding protein Astrin. SNM1B/Apollo interacts with Astrin through its conserved metallo-β-lactamase domain, and disruption of this interaction by point mutations results in a deficient prophase checkpoint. These findings suggest that SNM1B/Apollo and Astrin function together to enforce the prophase checkpoint in response to spindle stress.     

Human SNM1A suppresses the DNA repair defects of yeast pso2 mutants

Pso2/Snm1 plays a key role in the repair of DNA interstrand cross-links in yeast. Human cells possess three orthologues of Pso2; SNM1A, SNM1B/Apollo and SNM1C/Artemis. Studies using mammalian cells disrupted or depleted for these genes have yielded equivocal evidence that any of these is a true functional homologues of the yeast gene. Here we show that ectopic expression of only one of the three human orthologues, hSNM1A, effectively suppresses the sensitivity of yeast pso2 (snm1) disruptants to cross-linking agents. Two other phenotypes of the pso2 mutants are also partially rescued by ectopic expression of hSNM1A, namely the double-strand repair break defect observed during cross-link processing in pso2 cells, as well as the spontaneous intrachromatid recombination defect of pso2 msh2 double mutants. Finally, we show that recombinant hSNM1A is a 5'-exonuclease, as also recently reported for the yeast Pso2 protein. Together our data suggest that hSnm1A is a functional homologue of yeast Pso2/Snm1.     

DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation

The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways. In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown. We disrupted each of the SNM1 family genes in the chicken B-cell line DT40. Both SNM1A- and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin. SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair. SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-beta-lactamase) domain. PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci. Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells. These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair.     

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.     

Pso2 (SNM1) is a DNA structure-specific endonuclease

Many types of DNA structures are generated in response to DNA damage, repair and recombination that require processing via specialized nucleases. DNA hairpins represent one such class of structures formed during V(D)J recombination, palindrome extrusion, DNA transposition and some types of double-strand breaks. Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast. Pso2 (SNM1A in mammals) belongs to a small group of proteins thought to function predominantly during interstrand crosslink (ICL) repair. In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates. Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease. This activity was further shown to be required in vivo for repair of chromosomal breaks harboring closed hairpin ends. These findings provide the first evidence that Pso2 may function outside ICL repair and open the possibility that Pso2 may function at least in part during ICL repair by processing DNA intermediates including DNA hairpins or hairpin-like structures.     

The role of SNM1 family nucleases in etoposide-induced apoptosis

DNA double strand breaks (DSBs) induced by etoposide, an inhibitor of DNA topoisomerase II, are repaired mainly by non-homologous end joining (NHEJ). Unexpectedly, it was found that at high doses of etoposide, proteins involved in NHEJ, such as KU70/80, DNA-PKcs and ARTEMIS/SNM1C, trigger apoptosis rather than repair of DSBs. Because ARTEMIS is a member of the SNM1 protein family that includes SNM1A and APOLLO/SNM1B, this study examined whether SNM1A and/or APOLLO are also involved in etoposide-induced apoptosis. Using SNM1A^−/− and APOLLO^−/− cells, it was found that both SNM1A and APOLLO participate in etoposide-induced apoptosis. Although cell viability monitored by MTT assay did not differ between SNM1A^−/−/APOLLO^−/−/ARTEMIS^−/−, SNM1A^−/−/APOLLO^−/−, and single gene knockout cells, DNA fragmentation monitored by TUNEL assay differed between these cells, suggesting that the three SNM1 family nucleases function independently, at least during the induction of apoptotic DNA fragmentation.     

RAD18-dependent Recruitment of SNM1A to DNA Repair Complexes by a Ubiquitin-binding Zinc Finger

SNM1A is a member of the SNM1 family of nucleases required for cellular processing of interstrand DNA crosslinks (ICLs). Little is known about the molecular function of SNM1A, in terms of its recruitment to ICL lesions or its DNA damage processing activity. Here we show that SNM1A contains a functional PIP box (PCNA-interacting protein box) and a UBZ (ubiquitin binding zinc finger), required for assembly of SNM1A into nuclear focus. Moreover, RAD18-dependent monoubiquitination of PCNA is required for Mitomycin C and Ultraviolet Light inducible SNM1A nuclear focus assembly. Taken together, our results identify a novel RAD18-PCNA(Ub)-SNM1A pathway required for nuclear focus formation and ICL resistance.     

Hypoxia-inducible factor-1α regulates the expression of nucleotide excision repair proteins in keratinocytes

The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1α (HIF-1α) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1α increased XPC mRNA expression due to competition between HIF-1α and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1α protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1α also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1α is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1α downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1α in the repair of UVB-induced DNA damage.     

Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA–RPA and XPC–RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSβ were sensitive to psoralen ICLs. To further investigate the role of MutSβ in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSβ and NER proteins with Tdp-ICLs. We found that MutSβ bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSβ interacted with XPA–RPA or XPC–RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair.     

Non-erythroid alpha spectrin prevents telomere dysfunction after DNA interstrand cross-link damage

Telomere integrity is critical for telomere function and genomic stability. We previously demonstrated that non-erythroid α-spectrin (αIISp) is present in mammalian cell nuclei where it is important in repair of DNA interstrand cross-links (ICLs) and chromosome stability. We now demonstrate that αIISp is also important for telomere maintenance after ICL damage. It localizes to telomeres in S phase after ICL damage where it has enhanced association with TRF1 and TRF2 and is required for recruitment of the ICL repair protein, XPF, to damage-induced foci at telomeres. In telomerase-positive normal cells depleted of αIISp by siRNA or in Fanconi anemia, complementation group A (FA-A) cells, where αIISp levels are 35–40% of normal, ICL damage results in failure of XPF to localize to telomeres, markedly increased telomere dysfunction-induced foci, followed by catastrophic loss of telomeres. Restoration of αIISp levels to normal in FA-A cells corrects these deficiencies. Our studies demonstrate that αIISp is critical for repair of DNA ICLs at telomeres, likely by facilitating the recruitment of repair proteins similar, but not identical, to its proposed role in repair of DNA ICLs in genomic DNA and that this function in turn is critical for telomere maintenance after DNA ICL damage.     

The telomeric protein SNM1B/Apollo is required for normal cell proliferation and embryonic development

Conserved metallo β‐Lactamase and β‐CASP (CPSF‐Artemis‐Snm1‐Pso2) domain nuclease family member SNM1B/Apollo is a shelterin‐associated protein that localizes to telomeres through its interaction with TRF2. To study its in vivo role, we generated a knockout of SNM1B/Apollo in a mouse model. Snm1B/Apollo homozygous null mice die at birth with developmental delay and defects in multiple organ systems. Cell proliferation defects were observed in Snm1B/Apollo mutant mouse embryonic fibroblasts (MEFs) owing to high levels of telomeric end‐to‐end fusions. Deficiency of the nonhomologous end‐joining (NHEJ) factor Ku70, but not p53, rescued the developmental defects and lethality observed in Snm1B/Apollo mutant mice as well as the impaired proliferation of Snm1B/Apollo‐deficient MEFs. These findings demonstrate that SNM1B/Apollo is required to protect telomeres against NHEJ‐mediated repair, which results in genomic instability and the consequent multi‐organ developmental failure. Although Snm1B/Apollo‐deficient MEFs exhibited high levels of apoptosis, abrogation of p53‐dependent programmed cell death did not rescue the multi‐organ developmental failure in the mice.     

The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair

The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein–protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.     

SNM1A acts downstream of ATM to promote the G1 cell cycle checkpoint

We have shown previously that SNM1A colocalizes with 53BP1 at sites of double-strand breaks (DSBs) induced by IR, and that these proteins interact with or without DNA damage. However, the role of SNM1A in the DNA damage response has not been elucidated. Here, we show that SNM1A is required for an efficient G1 checkpoint arrest after IR exposure. Interestingly, the localization of SNM1A to sites of DSBs does not require either 53BP1 or H2AX, nor does the localization of 53BP1 require SNM1A. However, the localization of SNM1A does require ATM. Furthermore, SNM1A is shown to be a phosphorylation substrate of ATM in vitro, and to interact with ATM in vivo particularly after exposure of cells to IR. In addition, in the absence of SNM1A the activation of the downstream ATM target p53 is reduced. These findings suggest that SNM1A acts with ATM to promote the G1 cell cycle checkpoint.     

RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.     

SLX4 dampens MutSα-dependent mismatch repair

The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSβ (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSβ and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.     

Differential Roles for DNA Polymerases Eta,Zeta, and REV1 in Lesion Bypass of Intrastrand versus Interstrand DNA Cross-Links

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Polη), REV1, and zeta (Polζ) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polζ are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polζ facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Polη, whereas RAD18 plus Polη, REV1, and Polζ are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.Maintenance of genomic integrity involves the activation of cell cycle checkpoints coupled with DNA repair. Despite these sophisticated mechanisms to remove DNA lesions prior to DNA replication, replication forks may inevitably encounter nonrepaired lesions that block high fidelity polymerases, potentially leading to replication fork instability, gaps in replicated DNA, and the generation of DNA double-strand breaks (DSBs). In order to preserve replication fork stability by allowing replication through polymerase blocking lesions, template DNA containing a damaged base or abasic site can be replicated through the actions of specialized translesion DNA synthesis (TLS) polymerases (61). A key event in the regulation of TLS is the monoubiquitination of PCNA, a homotrimeric protein that functions as an auxiliary factor for DNA polymerases (28, 31, 57, 60). The RAD6 (E2)-RAD18 (E3) complex specifically monoubiquitinates PCNA on Lys-164 in response to replication fork stalling. This event is thought to operate as a molecular switch from normal DNA replication to the TLS pathway based on the observations that association of Y-family TLS polymerases with monoubiquitinated PCNA is strengthened through the cooperative binding of one or more ubiquitin-binding domains (UBM or UBZ) plus a PCNA-interacting domain (6, 25).Extensive biochemical evidence suggests that replication through a large variety of lesions requires the sequential action of two TLS polymerases (44). The Y-family polymerase eta (Polη) plays a key role in the efficient and error-free bypass of cyclobutane pyrimidine (TT) dimers, one of the major lesions resulting from exposure to UV radiation (45). In contrast, Polη can only insert a nucleotide directly opposite other lesions and requires an additional TLS polymerase, such as Polζ, to extend beyond the insertion (45). Polζ is comprised of the REV3 catalytic subunit that shares homology with B-family polymerases plus the REV7 accessory subunit (34). Polζ is unusual compared to other TLS polymerases due to the fact that it is relatively efficient at extending beyond mispaired primer termini and nucleotides inserted opposite a variety of DNA lesions, although this may occur in a potentially mutagenic manner (45). Genetic evidence in yeast suggest that Polζ activity is regulated by the Y family REV1 polymerase (21). In addition to a UBM domain that directly interacts with monoubiquitinated PCNA, REV1 possesses an N-terminal BRCT motif that directly contacts PCNA and potentially other proteins (24, 25). In addition, REV1 possesses a unique protein interaction domain in its carboxy terminus that interacts with the Polζ accessory subunit, REV7, and other TLS polymerases, including Polη and the Polζ catalytic subunit, REV3 (1, 18, 23, 40, 58). The characterization of these protein-protein interaction domains has led to the proposal that REV1 facilitates polymerase switching from a polymerase that directly inserts a nucleotide opposite a damaged base and Polζ, which subsequently performs the extension step beyond the inserted nucleotide opposite the damaged base (21).In addition to facilitating direct lesion bypass and filling in postreplicative gaps in DNA, REV1 and Polζ may also play an important role in the repair of interstrand cross-links (46, 63). Deletion of REV1, REV3, or REV7 in chicken DT40 cells leads to remarkable hypersensitivity to a wide variety of genotoxic stresses, most notably cisplatin and other DNA cross-linking agents such as mitomycin C (MMC) (38, 41, 55, 56). The genetic epistasis observed between REV1, REV3, and the Fanconi anemia (FA) complementation group C (FANCC) gene for cisplatin sensitivity further implicates TLS in the interstrand cross-link repair pathway (38). Current models suggest that when two replication forks converge upon an interstrand cross-link, the MUS81-EME1 endonuclease recognizes and cleaves the resulting branched DNA structure by making an incision at one side of the interstrand cross-link creating a replication-associated DSB (26). The XPF-ERCC1 endonuclease uncouples the cross-linked cDNA strands by making an incision on the other side of the interstrand cross-link (37). Recent biochemical evidence suggests that Polζ performs DNA synthesis opposite the DNA strand containing the residual cross-link and this process may be necessary to prepare the daughter strand for subsequent homologous recombination repair of the replication-associated DSB (46).Agents that introduce intra- and interstrand cross-links are widely used in cancer chemotherapy, and thus understanding the means by which cells repair or cope with these lesions will be instrumental in identifying novel mechanisms leading to drug resistance and designing new agents refractory to DNA damage tolerance mechanisms. Polη, REV1, and Polζ have all been implicated in mediating TLS past cisplatin intrastrand cross-links since lowering their expression increases sensitivity and reduces cisplatin-induced mutagenesis in human cancer cells (2, 5, 12, 42, 62). Furthermore, biochemical and structural analyses of Polη identified this polymerase as being capable of efficiently inserting dCTP opposite the 3′dG of a 1,2-d(GpG) cisplatin intrastrand cross-link (3). Here, we demonstrate that RAD18, Polη, and REV1 all localized to sites of replication stress marked by PCNA and γ-H2AX foci after treatment of cells with cisplatin. However, REV1 focus formation is specifically dependent upon both RAD18 and a functional FA core complex, suggesting FA core proteins are also necessary for directing REV1 to cisplatin-induced stalled replication forks. In addition, depletion of RAD18, Polη, REV1, or Polζ proteins lead to the induction of cellular responses indicative of inefficient lesion bypass of cisplatin adducts. Unexpectedly, we found that REV1- or Polζ-depleted cells displayed a greater loss in cell viability and the accumulation of chromosome aberrations and failed to resolve DSBs after cisplatin treatment. These results lead us to hypothesize that REV1 and Polζ may be necessary for the repair of cisplatin interstrand cross-links in addition to performing lesion bypass of cisplatin intrastrand cross-links. In agreement with this concept, we found that REV1 and Polζ-depleted cells were uniquely hypersensitive to MMC, accumulated greater numbers of chromosome aberrations, and failed to resolve replication-associated DSBs induced by MMC treatment.Together our findings support a model where replicative bypass of cisplatin intrastrand cross-links requires cooperation of multiple TLS polymerases in mammalian cells and is triggered by PCNA monoubiquitination. Our results also provide evidence that REV1 and Polζ facilitate repair of interstrand cross-links in human cells, and this process is likely independent of PCNA monoubiquitination.