Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2

SN1 DNA methylating agents such as the nitrosourea N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases.     

Evaluation of cadmium genotoxicity in Lactuca sativa L. using nuclear microsatellites

Cadmium (Cd) is a non-essential element and is a widespread environmental pollutant. Exposure to cadmium can result in cytotoxic, carcinogenic and mutagenic effects. Mutagenesis is indicative of genetic instability and can be assayed using microsatellites. Microsatellites or simple sequence repeats (SSRs) are composed of tandem repeats of short sequence motifs (1–6 bp) that are polymorphic, mainly in the number of tandem repeated units. Therefore, chromosomic mutations like inversion, deletion or translocation and point mutations can be detected by this type of molecular marker. In this study we have evaluated the mutagenic/genotoxic effects of cadmium in lettuce (Lactuca sativa L.). Five-week-old lettuce plants grown in a modified Hoagland's medium were exposed for a further 14 days to a medium containing 100 μM Cd(NO₃)₂. Genomic DNA was extracted from lettuce leaves and roots, harvested at days 0, 1, 3, 7 and 14, and nine SSRs were tested, amplified and analysed to evaluate microsatellite instability (MSI). Mutagenic effects of cadmium on microsatellite DNA loci were assessed and no MSI was observed in the used markers.     

A PLAC8-containing protein from an endomycorrhizal fungus confers cadmium resistance to yeast cells by interacting with Mlh3p

Cadmium is a genotoxic pollutant known to target proteins that are involved in DNA repair and in antioxidant defence, altering their functions and ultimately causing mutagenic and carcinogenic effects. We have identified a PLAC8 domain-containing protein, named OmFCR, by a yeast functional screen aimed at identifying genes involved in cadmium resistance in the endomycorrhizal fungus Oidiodendron maius. OmFCR shows a remarkable specificity in mediating cadmium resistance. Both its function and its nuclear localization in yeast strictly depend on the interaction with Mlh3p, a subunit of the mismatch repair (MMR) system. Although proteins belonging to the PLAC8 family are widespread in eukaryotes, they are poorly characterized and their biological role still remains elusive. Our work represents the first report about the potential role of a PLAC8 protein in physically coupling DNA lesion recognition by the MMR system to appropriate effectors that affect cell cycle checkpoint pathways. On the basis of cell survival assays and yeast growth curves, we hypothesize that, upon cadmium exposure, OmFCR might promote a higher rate of cell division as compared to control cells.     

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.     

Cadmium inhibits the functions of eukaryotic MutS complexes

Exposure of yeast cells to low concentrations of cadmium results in elevated mutation rates due to loss of mismatch repair (MMR), and cadmium inhibits MMR activity in extracts of human cells. Here we show that cadmium inhibits both Msh2-Msh6- and Msh2-Msh3-dependent human MMR activity in vitro. This inhibition, which occurs at a step or steps preceding repair DNA synthesis, is observed for repair directed by either a 3' or a 5' nick. In an attempt to identify the protein target(s) of cadmium inhibition, we show that cadmium inhibition of MMR is not reversed by addition of zinc to the repair reaction, suggesting that the target is not a zinc metalloprotein. We then show that cadmium inhibits ATP hydrolysis by yeast Msh2-Msh6 but has no effect on ATPase hydrolysis by yeast Mlh1-Pms1. Steady state kinetic analysis with wild type Msh2-Msh6, and with heterodimers containing subunit-specific Glu to Ala replacements inferred to inactivate the ATPase activity of either Msh2 or Msh6, suggest that cadmium inhibits ATP hydrolysis by Msh6 but not Msh2. Cadmium also reduces DNA binding by Msh2-Msh6 and more so for mismatched than matched duplexes. These data indicate that eukaryotic Msh2-Msh3 and Msh2-Msh6 complexes are targets for inhibition of MMR by cadmium, a human lung carcinogen that is ubiquitous in the environment.     

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers MSH2 and Cdt2 protein-dependent degradation of the cell cycle and mismatch repair (MMR) inhibitor protein p21Waf1/Cip1

p21(Waf1/Cip1) protein levels respond to DNA damage; p21 is induced after ionizing radiation, but degraded after UV. p21 degradation after UV is necessary for optimal DNA repair, presumably because p21 inhibits nucleotide excision repair by blocking proliferating cell nuclear antigen (PCNA). Because p21 also inhibits DNA mismatch repair (MMR), we investigated how p21 levels respond to DNA alkylation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which triggers the MMR system. We show that MNNG caused rapid degradation of p21, and this involved the ubiquitin ligase Cdt2 and the proteasome. p21 degradation further required MSH2 but not MLH1. p21 mutants that cannot bind PCNA or cannot be ubiquitinated were resistant to MNNG. MNNG induced the formation of PCNA complexes with MSH6 and Cdt2. Finally, when p21 degradation was blocked, MNNG treatment resulted in reduced recruitment of MMR proteins to chromatin. This study describes a novel pathway that removes p21 to allow cells to efficiently activate the MMR system.     

Cadmium inhibits human DNA mismatch repair in vivo

The heavy metal cadmium (Cd) is a human carcinogen that inhibits DNA repair activities. We show that DNA mismatch repair (MMR)-mediated cell cycle arrest after alkylation damage is suppressed by exposure to Cd and that this effect is reversed by preincubation with excess of zinc (Zn). We show that Cd-mediated inactivation of MMR activity is not caused by disruption of complex formation between the MMR proteins hEXO1-hMutS alpha and hEXO1-hMutL alpha nor does Cd inhibit 5'-exonuclease activity of hEXO1 in vitro. Thus, our studies show that exposure of human cells to Cd suppresses MMR activity, a repair activity known to play an important role in colon cancer and that this effect can be reversed by Zn treatment.     

The cell cycle and DNA mismatch repair

The DNA mismatch repair (MMR) pathway contributes to the fidelity of DNA synthesis and recombination by correcting mispaired nucleotides and insertion/deletion loops (IDLs). We have investigated whether MMR protein expression, activity, and subcellular location are altered during discrete phases of the cell cycle in mammalian cells. Two distinct methods have been used to demonstrate that although physiological MMR protein expression, mismatch binding, and nick-directed MMR activity within the nucleus are at highest levels during S phase, MMR is active throughout the cell cycle. Despite equal MMR nuclear protein concentrations in S and G(2) phases, mismatch binding and repair activities within G(2) are significantly lower, indicating a post-translational decrease in MMR activity specific to G(2). We further demonstrate that typical co-localization of MutSalpha to late S phase replication foci can be disrupted by 2 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This concentration of MNNG does not decrease ongoing DNA synthesis nor induce cell cycle arrest until the second cell cycle, with long-term colony survival decreased by only 24%. These results suggest that low level alkylation damage can selectively disrupt MMR proofreading activity during DNA synthesis and potentially increase mutation frequency within surviving cells.     

Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

Treatment with low concentrations of monofunctional alkylating agents induces a G2 arrest only after the second round of DNA synthesis in mammalian cells and requires a proficient mismatch repair (MMR) pathway. Here, we have investigated rapid alkylation-induced recruitment of DNA repair proteins to chromosomal DNA within synchronized populations of MMR proficient cells (HeLa MR) after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. Within the first hour, the concentrations of MutS alpha and PCNA increase well beyond their constitutive chromosomally bound levels and MutL alpha is newly recruited to the chromatin-bound MutS alpha. Remarkably, immunoprecipitation experiments demonstrate rapid association of these proteins on the alkylation-damaged chromatin, even when DNA replication is completely blocked. The extent of association of PCNA and MMR proteins on the chromatin is dependent upon the concentration of MNNG and on the specific type of replication block. A subpopulation of the MutS alpha-associated PCNA also becomes monoubiquitinated, a known requirement for PCNA to interact with translesion synthesis (TLS) polymerases. In addition, chromatin-bound SMC1 and NBS1 proteins, associated with DNA double-strand-breaks (DSBs), become phosphorylated within 1-2h of exposure to MNNG. However, these activated proteins are not co-localized on the chromatin with MutS alpha in response to MNNG exposure. PCNA, MutS alpha/MutL alpha and activated SMC1/NBS1 remain chromatin-bound for at least 6-8h after alkylation damage. Thus, cells that are exposed to low levels of alkylation treatment undergo rapid recruitment to and/or activation of key proteins already on the chromatin without the requirement for DNA replication, apparently via different DNA-damage signaling pathways.     

Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: The role of Grx2 under cadmium exposure

Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 μM CdSO₄, showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.     

MutS inhibits RecA-mediated strand transfer with methylated DNA substrates

DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O⁶-methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O⁶-methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecA-mediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentration-dependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O⁶-methylguanine residues in methylated DNA by HPLC-MS/MS and 5–10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system.     

Single-molecule analysis reveals the kinetics and physiological relevance of MutL-ssDNA binding

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (K_D = 29 nM) while it dramatically decreases above 100 mM (K_D>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.     

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.     

Formation and persistence of N7-methylguanine DNA adducts in the target pyloric tissue following chronic exposure to N-methyl-N'-nitro-N-nitrosoguanidine

Outbred 7-week old male Wistar rats were exposed for 21 days to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinking water and N7-methyl deoxyguanosine 3'-monophosphate (N7-MedGp) levels in DNA from the pyloric mucosa (target tissue) and white blood cells (wbc: non-target tissue) were determined by ³²P-postlabelling. Exposure to MNNG resulted in the non-linear, dose-related formation of N7-medGp in both tissues. Adduct levels in the pyloric mucosa were determined to be 1058, 5.4 and 1.1 μmole N7-medGp mole^-1 deoxyguanosine 3'-monophosphate (dGp) after exposure to 4.1, 0.62 and 0.006 mg MNNG kg^-1 day^-1 respectively whereas adduct levels in the wbc DNA were lower at 5.2, 0.52 and 0.68 μmoles N7-medGp mole^-1 dGp after exposure to 4.1, 0.62 and 0.062 mg MNNG kg^-1 day^-1 respectively. In addition, the persistence of N7-medGp was investigated. Loss of adduct occurred rapidly, with a decrease of 87 and 97% respectively in target tissue and wbc DNA by 48 h after cessation of 4.1 mg MNNG kg^-1 day^-1 exposure; 14 days post-MNNG treatment, however, N7-medGp was still detectable (0.46 μmole N7-medGp mole^-1 dGp) in pyloric mucosal DNA. The quantitation of N7-medGp after exposure to low doses of carcinogen, i.e. 0.006 mg MNNG kg^-1 day^-1, approaching environmentally relevant levels has not been previously reported, and indicates that the ³²P-postlabelling assay developed here possesses sufficient sensitivity to quantitate N7- medGp in human DNA arising from environmental exposure to methylating agents.     

Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: the role of Grx2 under cadmium exposure

Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 muM CdSO(4), showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.     

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G₂/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.     

Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1

In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutSα) and hMSH2/hMSH3 (hMutSβ) are the mismatch recognition factors of the mismatch repair pathway. hMutSα has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutSα and hMutLα. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutSα binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutSα. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.     

Interplay of DNA repair pathways controls methylation damage toxicity in Saccharomyces cerevisiae

Methylating agents of S(N)1 type are widely used in cancer chemotherapy, but their mode of action is poorly understood. In particular, it is unclear how the primary cytotoxic lesion, O(6)-methylguanine ((Me)G), causes cell death. One hypothesis stipulates that binding of mismatch repair (MMR) proteins to (Me)G/T mispairs arising during DNA replication triggers cell-cycle arrest and cell death. An alternative hypothesis posits that (Me)G cytotoxicity is linked to futile processing of (Me)G-containing base pairs by the MMR system. In this study, we provide compelling genetic evidence in support of the latter hypothesis. Treatment of 4644 deletion mutants of Saccharomyces cerevisiae with the prototypic S(N)1-type methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) identified MMR as the only pathway that sensitizes cells to MNNG. In contrast, homologous recombination (HR), postreplicative repair, DNA helicases, and chromatin maintenance factors protect yeast cells against the cytotoxicity of this chemical. Notably, DNA damage signaling proteins played a protective rather than sensitizing role in the MNNG response. Taken together, this evidence demonstrates that (Me)G-containing lesions in yeast must be processed to be cytotoxic.     

Exonuclease 1 (Exo1) is required for activating response to SN1 DNA methylating agents

S_N1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O⁶ position of guanine (O⁶meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O⁶meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the S_N1 methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to S_N1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.