Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Jul 27, 2010; 107(30): 13384–13389.
Published online Jul 12, 2010. doi:  10.1073/pnas.1008589107
PMCID: PMC2922181

PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance


The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2−/− mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.

Keywords: DNA mismatch repair, B cell lymphoma, class switch recombination, somatic hypermutation, spermatogenesis

PMS2 is an essential component of DNA mismatch repair (MMR) complexes, which play an important role in maintaining genetic stability. MMR functions primarily in the detection and repair of mismatched bases that result from erroneous replication, and also plays important roles in DNA damage response, genetic recombination, control of meiotic progression, and generation of antibody diversity (1). During MMR, heterodimeric MutS homolog (MSH) complexes, consisting of either MSH2 and MSH6 (MutSα) or MSH2 and MSH3 (MutSβ), bind to mismatched bases. On binding, a conformational change in the MutS complexes activates downstream events and leads to the recruitment of MutL homolog (MLH) complexes consisting of either MLH1 and PMS2 (MutLα) or MLH1 and MLH3 (MutLγ). MutLα interacts with the MutS complexes via MLH1, and this interaction is essential for the coordination of downstream repair events, including excision of the mismatch carrying strand and its resynthesis (2, 3).

Important insights into the role of MutLα in MMR came from biochemical studies of reconstituted human MMR (4, 5), which showed that PMS2 is a latent endonuclease that introduces additional nicks into the discontinuous DNA strand of nicked heteroduplex substrates. This activity is dependent on the integrity of a highly conserved metal-binding motif found in many MutL homologs, including MLH3. MutLα appears to play an essential role in 3′-directed MMR, where the bias for incision on the distal side of the mismatch results in a 5′ entry site for MutSα-activated Exo1. For 5′-directed MMR, however, several studies in both the reconstituted human MMR system and MutLα-deficient cells have shown that MutLα is not essential, and that a purified system consisting of MutSα, Exo1, and RPA is sufficient to support 5′-directed MMR (6, 7). Studies in yeast have further delineated roles of the PMS2 endonuclease activity in DNA repair, recombination, and the DNA damage response (5, 8, 9).

Pms2−/− mice (10) showed an increase in base substitution and insertion/deletion mutation frequencies at both a reporter locus and microsatellite sequences, with a higher proportion of frameshift mutations compared with other MMR knockout models, such as Msh2−/− and Mlh1−/− mice (11, 12). As a result, Pms2−/− mice developed lymphomas and sarcomas (13). Moreover, Pms2−/− male mice are sterile, suggesting an important role for PMS2 in spermatogenesis. PMS2 also plays a role in class switch recombination (CSR) of Ig genes, because Pms2−/− mice (14, 15) and patients with deleterious homozygous mutations in PMS2 (16) have a significant reduction in isotype switching. Despite numerous reports (1619), however, the role of PMS2 in somatic hypermutation (SHM), if any, has not been resolved.

To determine the significance of the PMS2 endonuclease for these processes, we generated a mouse line with an E702K missense mutation in the metal-binding motif of PMS2. This mutation corresponds to the E705K mutation in human PMS2 that completely inactivates the endonuclease activity, and also has been found in a patient with Turcot syndrome (4). The analysis of Pms2E702K mice (termed Pms2EK) showed that the loss of this activity leads to a dramatic increase in genomic mutation frequencies and tumor incidence, and a significant decrease in CSR of the Ig locus. Strikingly, Pms2EK/EK male mice were fertile, as opposed to the sterile Pms2−/− male mice, indicating that the endonuclease function of PMS2 is dispensable in spermatogenesis and that an unknown function of the protein is required for this biological process.

Results and Discussion

Generation of Pms2EK/EK Mutant Mice.

To generate Pms2EK mutant mice, a gene-targeting vector was designed that introduces the murine equivalent of the human E705K mutation (E702K) into exon 12 of the mouse gene (Fig. 1Aand Fig. S1). This mutation disrupts the DQHA(X)2E(X)4E metal-binding motif, which is essential for the endonuclease function of PMS2 (4). RT-PCR and qPCR confirmed that the mutant allele was expressed at WT RNA levels in both mouse embryonic fibroblasts (MEFs) and primary B cells of Pms2EK/EK mice (Fig. 1 B and C). The presence of PMS2E702K protein in Pms2EK/EK mice was confirmed in splenic tissue by Western blot analysis (Fig. 1D); however, expression was reduced to ≈25% compared with that seen in WT mice. Similar results were obtained in brain, testis, and MEF cell lines. The reduced expression of PMS2E702K protein in Pms2EK/EK mice might be due to increased degradation of the mutant protein and is in line with previous results showing that transfecting Pms2−/− MEFs with hPMS2E705K leads to lower protein levels compared with cells transfected with WT hPMS2 (8). Consistent with previous studies (20), MLH1 protein levels were not affected by the reduced level of PMS2E702K protein (Fig. 1D), indicating either that sufficient mutant PMS2E702K protein is present to form a stable complex with MLH1 (in Pms2EK/EK mice) or that MLH1 is stabilized by formation of complexes with other proteins, such as MLH3 or PMS1. Similarly, MLH3 protein levels were not affected in any of the Pms2 genotypes (Fig. 1D).

Fig. 1.
Generation of Pms2EK/EK mice. (A) PMS2 domains and location of E702K. (B) RT-PCR analysis of total RNA from MEFs. The PCR product was digested with SacII to reveal specific patterns for all three Pms2 genotypes. (C) q-PCR analysis of total RNA from MEFs ...

Defective MMR in Pms2EK/EK Cells.

To determine the effect of the PMS2E702K mutation on MMR, we tested extracts from Pms2EK/EK, Pms2+/EK, Pms2−/−, and WT MEF cells for their ability to promote repair of heteroduplexes containing a G-T mismatch and a single-stranded nick located either 5′ or 3′ to the mismatched base. No MMR activity was detected in Pms2EK/EK or Pms2−/− extracts, whereas robust repair was seen in WT cells (Fig. 2A). These results demonstrate that the endonuclease function of PMS2 is essential for MMR in cell extracts. In contrast to previous biochemical studies showing that MutLα is not essential for 5′-directed MMR in a system using purified protein (6, 7), our results indicate that the endonuclease activity is essential in both 5′- and 3′-directed MMR in whole cell extracts.

Fig. 2.
MMR is impaired and leads to genomic instability in Pms2EK/EK mice. (A) MMR in whole cell extracts of MEFs. Repair was quantified from three independent experiments. (B) MSI analysis. The percent instability was determined by the number of unstable alleles ...

It has been reported that low levels of human WT PMS2, but not high levels of the E705K mutant protein, can restore MMR in Pms2−/− mouse cells (9). To further confirm that the repair deficiency in Pms2EK/EK extracts was caused by loss of endonuclease activity and not by the reduced level of mutant PMS2E702K protein, we added decreasing amounts of WT human MutLα protein to Pms2−/− cell extracts. Significant MMR activity was still detected when WT human MutLα levels were reduced to levels comparable to the MutLα levels observed in Pms2EK/EK cells (Table S1). The human and mouse PMS2 proteins share a high homology in their amino acid sequence, and the observation that the WT human protein can efficiently reconstitute MMR in Pms2−/− mouse cells further demonstrates that PMS2 functions are conserved. Thus, our results strongly suggest that the repair-deficient phenotype of Pms2EK/EK mice is specifically caused by mutation of the endonuclease domain.

Genomic Instability in Pms2EK/EK Mutant Mice.

The loss of PMS2 leads to increased microsatellite instability (MSI) in the genome of Pms2−/− mice (13). To study whether this increase in MSI was specifically caused by the loss of the endonuclease function, we analyzed MSI at two dinucleotide markers in mouse genomic DNA. At both markers, Pms2EK/EK mice displayed elevated levels of unstable alleles compared with WT mice: 12% versus 0.8% for marker D7Mit91 and 19% versus 5% for marker D17Mit123 (Fig. 2B). These MSI levels were similar to those observed in Pms2−/− mice (14% and 17%, respectively) and slightly lower than those reported in Mlh1−/− mice (15% and 28%, respectively) (21). Pms2+/EK mice showed no increase in MSI.

To investigate the effect of the loss of PMS2 endonuclease function on mutagenesis in more detail, we determined the mutation rate at the cII reporter locus in 10-wk-old Pms2EK/EK, Pms2+/EK, Pms2−/−, and WT littermates. DNA was isolated from spleen, liver, and small intestine. In all three organs, mutation frequencies were significantly increased in Pms2EK/EK mice compared with WT and Pms2+/EK mice (Fig. 2C). The mutation frequencies tended to be slightly higher in Pms2−/− mice than in Pms2EK/EK mice, but the differences were not significant. As reported previously for Pms2−/− mice (22), there was a greater increase in the number of insertions and deletions in Pms2EK/EK mice compared with WT mice (Fig. 2D). Pms2EK/EK and Pms2−/− mice showed a significant increase in mutations at A:T in spleen and small intestine (P < 0.001) (Fig. 2E). A similar increase in A:T mutations was described previously in T cells of Pms2−/− mice (23). Mutations at A:T could have been generated by the error-prone DNA polymerase η (24). Interestingly, an interaction between MutLα and Pol η has been reported recently (25), and it has been suggested that this interaction could facilitate error-free DNA replication by repairing the mismatches introduced by Pol η. The increase in mutations at A:T seen in Pms2EK/EK and Pms2−/− mice is consistent with this notion.

Reduced Survival and Cancer Phenotype in Pms2EK/EK Mutant Mice.

Similar to Pms2−/− mice (n = 28), Pms2EK/EK mice (n = 39) had significantly lower survival compared with age-matched Pms2+/EK mice (n = 27) and WT mice (n = 28) (P < 0.001) (Fig. 2F). This difference in survival was due primarily to increased predisposition to lymphomas (90%; 26/29 mice), mostly B cell lymphomas (83%; 20/24 mice) (Table S2), although other tumors also occurred, including sarcoma (5/29 mice), hepatic adenoma (2/29 mice), hemangioma (1/29 mice), intestinal adenocarcinoma (1/29 mice), and uterine adenocarcinoma (1/29 mice). Compared with Mlh1−/− mice, which have a median survival time of 7 mo (13, 26), the reduced survival of Pms2EK/EK and Pms2−/− mice is more moderate (12 and 13 mo, respectively). This suggests the existence of a partial MMR defect in these mice in vivo, possibly due to either functional redundancy with other MutL complexes, such as MLH1-MLH3, or an abundance of preexisting DNA nicks that occur during normal DNA metabolic processes, such as replication.

The B cell lymphoma phenotype in Pms2EK/EK and Pms2−/− mice differs from that in Msh2−/− (T cell lymphoma) (27) and Mlh1−/− (B and T cell lymphoma) (26) mice, and indicates that PMS2 endonuclease activity has an important tumor-suppressor function in the B cell lineage. The similarities in the survival and tumor spectra of Pms2EK/EK and Pms2−/− mice suggest that endonuclease activity is the major function of PMS2 required for prevention of genome instability and tumorigenesis.

Pms2EK/EK Mutant Mice Show a Decrease in ex Vivo CSR.

To examine whether inactivation of the endonuclease function of PMS2 affects CSR, we purified splenic B cells from Pms2EK/EK, Pms2−/−, and WT mice and stimulated them ex vivo to induce switching from IgM to IgG3, IgG1, or IgG2b. Pms2−/− mice showed a ~40–70% reduction in switching efficiencies compared with WT mice (P < 0.001) (Fig. 3A). This result is consistent with previous reports on truncated forms of PMS2 (16) and on Pms2−/− mice (14, 15), which showed significant deficiencies in CSR to all studied isotypes. Compared with WT mice, Pms2EK/EK mice also exhibited a ~40–60% reduction in switching (P < 0.001), but this decrease was not significantly different from that observed in Pms2−/− cells (P ≥ 0.12). The defect in CSR was due to Pms2EK/EK samples accumulating fewer cells that had switched at each cell division, not to a change in the doubling time of the stimulated B cells (Fig. 3B) or a decrease in the frequency of B220+PNAhi germinal center B cells in the spleen (Fig. 3C). These findings suggest that PMS2 endonuclease activity, as opposed to other protein functions, does not merely delay the switching process, but rather is the major contributor to PMS2 function during CSR.

Fig. 3.
Reduced class switching in Pms2EK/EK mice. (A) Pms2EK/EK (n = 5) and Pms2−/− (n = 3) mice assayed in three independent experiments showed a similar decrease in the relative switching to IgG3, IgG1, and IgG2b compared with their WT littermates ...

Pms2−/− and Pms2EK/EK Mice Show Differences in Microhomology-Mediated Mechanisms at Sμ–Sγ3 Junctions.

It has been suggested that in the absence of the MLH1–PMS2 complex, an alternative end-joining pathway based on long microhomologies at the junction might provide stability to the synaptic structure during CSR (14, 28). Indeed, our Pms2−/− mice had Sμ–Sγ3 junctional DNA segments with extended microhomology (≥5 nts) and a longer average length compared with WT mice (5.07 ± 1.4 vs. 2.6 ± 0.44; P = 0.04) (Table 1). Abnormal formation of switch junctions also has been identified in truncated forms of human PMS2 (16). In contrast, although we detected a tendency toward a preferential use of blunt junctions in Pms2EK/EK mice compared with WT mice (Table 1), we found no significant differences in the relative frequencies of the different types of junctions (i.e., blunt junctions, microhomologies, or insertions) or in the average length of microhomology (2.54 ± 0.53 vs. 2.6 ± 0.44; P = 0.47) between the Pms2EK/EK and the WT mice (Table 1). These findings indicate that the presence of the PMS2 protein, but not its endonuclease activity, affects the microhomology-mediated end-joining mechanisms of CSR, suggesting multiple roles for PMS2 during CSR.

Table 1.
Sμ–Sγ3 junctions in stimulated Pms2+/+, Pms2−/−, and Pms2EK/EK splenic B cells

Somatic Hypermutation Is Not Impaired in Pms2−/− or Pms2EK/EK Mice.

Although the present study and previous work have shown that PMS2 plays a role in postreplicative MMR and in the repair of double-stranded DNA breaks in Ig switching regions, deficiency of the protein has a modest or no effect during SHM of the Ig locus (1619). Considering the possibility that other factors (e.g., MLH3 or apurinic/apyrimidinic endonuclease) might efficiently compensate for the absence of PMS2 and fulfill the endonucleolytic requirements of V-region hypermutation, we investigated SHM in our mutant mice. Pms2EK/EK, Pms2−/− and WT mice were immunized with NP30-CGG, and splenic B cells were analyzed for the pattern of somatic mutations in the rearranged VH186.2 gene. Based on unique mutations and correcting for base composition of the VH186.2 sequence, we found no significant differences in the overall mutation frequency among WT, Pms2−/−, and Pms2EK/EK mice (P ≥ 0.41) (Table 2). The frequencies of the different types of substitutions (P ≥ 0.11) and of transversions or transitions were similar (P ≥ 0.35) in the Pms2−/− and Pms2EK/EK mice and not significantly different from those in their WT littermates. This indicates that neither the presence of the protein nor the endonuclease activity of PMS2 is essential for the accumulation of mutations during SHM. Consistent with this, we found no difference among genotypes in the frequency of dinucleotide substitutions (P ≥ 0.84) or a decreased ratio of mutations in A as opposed to T (P ≥ 0.45). In fact, Pms2−/− and Pms2EK/EK mice retained the strand bias signature previously attributed to the error-prone polymerase Pol η (29, 30), in which mutations at A sites and WA motifs exceed mutations at T sites and TW motifs. Furthermore, the overall mutation frequencies of the hotspots for AID (WRC/GYW) and Pol η (WA/TW), which are major hallmarks of the SHM process, were similar in WT, null, and mutant mice (P ≥ 0.17). The lack of effect of the mutant PMS2E702K protein on the frequency and characteristics of A:T mutations in the variable region gene, even in the presence of increased A:T mutations in the cII reporter gene, confirms that PMS2 does not play a significant role in SHM.

Table 2.
Unique mutation frequency analysis of the VH186.2 region

PMS2 Endonuclease Is Not Required for Spermatogenesis.

Pms2−/− male mice are sterile (10), indicating that PMS2 plays an important role in meiotic processes. Strikingly, in the present study, Pms2EK/EK male and female mice were fertile, indicating normal meiotic progression. Pms2EK/EK testes contained normal seminiferous epithelium with mature spermatozoa in the lumen, indicating completion of spermatogenesis (Fig. 4A). In contrast, the seminiferous tubules of Pms2−/− mice were severely depleted in spermatogenic cells, and mature sperm cells were abnormal, with misshaped heads and truncated or coiled flagella (Fig. 4B, Bottom Right). Testis size in Pms2−/− mice was about 70% of that in WT littermates, whereas testis size in Pms2EK/EK mice was similar to that in WT littermates (Fig. 4B, Left and Top Right). Progression through meiotic prophase I appeared to be normal in all three genotypes, however (Fig. 4C). These results indicate that the endonuclease function of PMS2 does not play an essential role in meiosis, and that the presence of the protein, whether in a scaffolding function or another unknown function, is essential for spermatogenesis. PMS2 functions in the response to DNA damage in mitotic cells (31), and it is possible that the protein also contributes to checkpoint-signaling pathways during premeiotic S-phase and spermatogenesis.

Fig. 4.
Testis morphology in male mice. (A) H&E staining of testis sections from 10-wk-old WT, Pms2EK/EK, and Pms2−/− mice. Note the regions of degeneration in the testis of Pms2−/− males. In WT and Pms2EK/EK mice, the ...


Our analysis of Pms2EK mutant mice reveals essential roles of the PMS2 endonuclease activity in mutation avoidance and tumor suppression in mammalian tissues. It also demonstrates that the PMS2EK mutation acts as a separation-of-function mutation and exerts distinct effects on different biological processes, including B cell antibody diversification and spermatogenesis. Although both CSR and SHM are initiated by the same AID-dependent dU:G mismatch, PMS2 endonucleolytic or scaffolding functions are not essential for SHM, but significantly contribute to CSR. In fact, although PMS2 endonuclease activity is essential for MutLα-dependent processing of CSR intermediates, the PMS2 protein clearly provides additional functions in the process. Although it is possible that the differences between the WT and Pms2EK/EK mutant mice are due in part to the decreased abundance of PMS2, the presence of longer microhomologies at CSR junctions in Pms2−/− mice, but not in Pms2EK/EK mice, suggests that there is sufficient PMS2 protein in Pms2EK/EK mice to provide scaffolding and to preclude the need for long microhomologies during CSR. The importance of PMS2 as a scaffolding protein, even at reduced cellular levels, is further highlighted by the finding that Pms2EK/EK male mice have no obvious defects in spermatogenesis. The identity of the meiotic partners of PMS2 other than MLH1 is currently unknown, and their identification will provide important insight into the mechanisms that control meiotic progression and/or sperm differentiation.

Materials and Methods

Generation and Analysis of Pms2E702K Mice.

Pms2E702K mice were created by a knock-in gene-targeting strategy (Fig. S1). All protocols involving animals were approved by the Albert Einstein College of Medicine's Animal Care and Use Committee in accordance with the U.S. Public Health Service's Animal Welfare Policy. RT-PCR using primers located in exon 12 (5′-CGATGTTTGCAGAGATGGAG-3′) and exon 14 (5′-TAGCCCTTTCAGTGACTGGAG-3′) of Pms2 was done on total RNA isolated from MEF cell lines. Relative quantification of Pms2 transcripts was performed on cDNA from resting splenic B cells and MEFs by real-time PCR using primers 5′-AACCGAAGGCGTGAGTACAG-3′ and 5′-CACAGCGGTGCTTAAACTGA-3′. For Western blot analysis, equal amounts of total cellular protein from MEF and tissue extracts were separated on 3–8% NuPAGE gels (Invitrogen). Proteins were detected with anti-PMS2 (clone A16-4; BD PharMingen), anti-MLH1 (Bethyl Laboratories), or anti-MLH3 (a gift from Paula E. Cohen, Cornell University, Ithaca, NY). Bands were quantified using GeneTools software (Syngene).

Mismatch Repair Assay.

MEFs were cultured in DMEM high-glucose medium supplemented with 10% FBS (HyClone). Whole cell extracts were prepared as described previously (32), as were 5′ G-T heteroduplex and 3′ G-T heteroduplex DNAs (33, 34). MMR reactions using whole cell extracts were carried out following the method of Kadyrov et al. (32). Recombinant human MutLα used to complement the whole cell extracts of Pms2−/− and Pms2EK/EK MEFs was isolated as described previously (35).

Microsatellite Instability Analysis.

Tail DNA from six WT, six Pms2+/EK, six Pms2EK/EK, and six Pms2−/− mice was pooled and diluted to approximately one molecule per reaction. The microsatellite loci were amplified and analyzed as described previously (36).

cII Mutation Rate Analysis.

Mutations in genomic DNA from the spleen, liver, and small intestine of Pms2EK/EK mice crossed to Big Blue transgenic mice (37) were detected using the λ Select-cII Mutation Detection System for Big Blue Rodents (Stratagene). Mutation frequency was defined as the ratio of mutant plaques to the total number of plaques screened. The mutation spectrum for base substitutions was analyzed using the SHMTool Web server (38).

Analysis of Tumors and Survival.

Mice were observed until they became morbid or moribund. Tumors were removed and fixed in 10% buffered formalin. After being embedded in paraffin, sections were stained with H&E, anti-B220 (BD PharMingen), or anti-CD3 (Lab Vision). Statistical analysis of tumor incidence was done using SPSS version 16.0 (SPSS). The Kaplan-Meier method was used to compare curves for survival, with significance evaluated by two-sided log rank statistics.

Ex Vivo CSR and Switch Junction Analysis.

Splenic B cells were stimulated with 50 μg/mL of LPS (Sigma-Aldrich), to induce switching to IgG3; LPS plus 50 ng/mL of rIL-4 (R&D Systems), to induce switching to IgG1; or LPS plus 30 μg/mL of dextran sulfate (DS) (Sigma-Aldrich) to induce switching to IgG2b. After 4 d in culture, surface IgM and IgG were stained and analyzed by FACS. PCR amplification from genomic DNA, sequencing, and analysis of unique junctional Sμ–Sγ3 regions were performed as described previously (39).

Somatic Hypermutation Analysis.

Eight-week-old mice were immunized i.p. with (4-hydroxy-3-nitrophenyl)acetyl (NP)30-CGG (BioSearch Technologies) in alum (Pierce) and boosted 4 wk later. TRIzol extraction (Invitrogen) of RNA was prepared 7 d after the boost from splenic B cells. Then high-fidelity RT-PCR and nested PCR were performed to amplify VH186.2 joined to the IgG1 constant region, as described previously (39). Analysis of unique mutations was done with SHMTool (38). The experimental error rate was estimated 2.2 × 10−4 mutations/base by analysis of the Cγ1 segment. A significant bias of mutations from A exceeding mutations from T in the WT data was used for quality assessment of contaminating PCR hybrids, as suggested recently (30).

Analysis of Meiotic Prophase I.

Chromosome spreads were prepared as described previously (40) with modifications. After cells were dispersed on slides for 1 h, the slides were washed three times for 3 min each in 0.4% Kodak Photo-Flo 200/water and air-dried. Then the slides were washed in 0.4% Kodak Photo-Flo 200/PBS and 0.1% Triton X-100/PBS for 3 min each, blocked for 3 min in blocking solution (3% BSA, 0.05% Triton X-100, 10% goat serum in PBS), and incubated in an antibody against synaptonemal complex protein 3 (SYCP3, a gift from Paula E. Cohen, Cornell University, Ithaca, NY) for 1 h at 37 °C in a humid chamber. The slides were then washed, blocked, and incubated in a secondary antibody conjugated to fluorescein (Jackson Immunochemicals) for 30 min at 37 °C. Images were captured with an Olympus BX61 upright microscope with a CoolSNAP HQ camera (Photometrics) and IPLab acquisition software (BD).

Supplementary Material

Supporting Information:


We thank B. Jin for providing excellent technical assistance, Y. Zhang and R. Chahwan for providing additional technical support, and P. Cohen (Cornell University, Ithaca, NY) for generously providing the MLH3 and SYCP3 antibodies. This work was supported by a Rubicon Fellowship from the Netherlands Organization for Scientific Research (to J.M.M.v.O.), Postdoctoral Fellowship EX-2006-0732 from the Spanish Ministry of Education and Science (to S.R.), and National Institutes of Health Grants GM45190 (to P.M.), CA72649 and CA102705 (to M.D.S.), and CA76329 and CA93484 (to W.E.). M.D.S. is supported by the Harry Eagle Chair provided by the National Women's Division of the Albert Einstein College of Medicine. P.M. is an Investigator at the Howard Hughes Medical Institute.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1008589107/-/DCSupplemental.


1. Iyer RR, Pluciennik A, Burdett V, Modrich PL. DNA mismatch repair: Functions and mechanisms. Chem Rev. 2006;106:302–323. [PubMed]
2. Kunkel TA, Erie DA. DNA mismatch repair. Annu Rev Biochem. 2005;74:681–710. [PubMed]
3. Marsischky GT, Kolodner RD. Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA. J Biol Chem. 1999;274:26668–26682. [PubMed]
4. Kadyrov FA, Dzantiev L, Constantin N, Modrich P. Endonucleolytic function of MutLalpha in human mismatch repair. Cell. 2006;126:297–308. [PubMed]
5. Kadyrov FA, et al. Saccharomyces cerevisiae MutLa is a mismatch repair endonuclease. J Biol Chem. 2007;282:37181–37190. [PMC free article] [PubMed]
6. Genschel J, Modrich P. Mechanism of 5′-directed excision in human mismatch repair. Mol Cell. 2003;12:1077–1086. [PubMed]
7. Genschel J, Modrich P. Functions of MutLalpha, replication protein A (RPA), and HMGB1 in 5′-directed mismatch repair. J Biol Chem. 2009;284:21536–21544. [PMC free article] [PubMed]
8. Deschênes SM, et al. The E705K mutation in hPMS2 exerts recessive, not dominant, effects on mismatch repair. Cancer Lett. 2007;249:148–156. [PMC free article] [PubMed]
9. Erdeniz N, Nguyen M, Deschênes SM, Liskay RM. Mutations affecting a putative MutLalpha endonuclease motif impact multiple mismatch repair functions. DNA Repair (Amst) 2007;6:1463–1470. [PMC free article] [PubMed]
10. Baker SM, et al. Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis. Cell. 1995;82:309–319. [PubMed]
11. Andrew SE, et al. Mutagenesis in PMS2- and MSH2-deficient mice indicates differential protection from transversions and frameshifts. Carcinogenesis. 2000;21:1291–1295. [PubMed]
12. Yao X, et al. Different mutator phenotypes in Mlh1- versus Pms2-deficient mice. Proc Natl Acad Sci USA. 1999;96:6850–6855. [PMC free article] [PubMed]
13. Prolla TA, et al. Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair. Nat Genet. 1998;18:276–279. [PubMed]
14. Ehrenstein MR, Rada C, Jones AM, Milstein C, Neuberger MS. Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination. Proc Natl Acad Sci USA. 2001;98:14553–14558. [PMC free article] [PubMed]
15. Schrader CE, Edelmann W, Kucherlapati R, Stavnezer J. Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes. J Exp Med. 1999;190:323–330. [PMC free article] [PubMed]
16. Péron S, et al. Human PMS2 deficiency is associated with impaired immunoglobulin class switch recombination. J Exp Med. 2008;205:2465–2472. [PMC free article] [PubMed]
17. Cascalho M, Wong J, Steinberg C, Wabl M. Mismatch repair co-opted by hypermutation. Science. 1998;279:1207–1210. [PubMed]
18. Di Noia JM, Neuberger MS. Molecular mechanisms of antibody somatic hypermutation. Annu Rev Biochem. 2007;76:1–22. [PubMed]
19. Saribasak H, Rajagopal D, Maul RW, Gearhart PJ. Hijacked DNA repair proteins and unchained DNA polymerases. Philos Trans R Soc Lond. 2009;364:605–611. [PMC free article] [PubMed]
20. Chen PC, et al. Contributions by MutL homologues Mlh3 and Pms2 to DNA mismatch repair and tumor suppression in the mouse. Cancer Res. 2005;65:8662–8670. [PubMed]
21. Wei K, et al. Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility. Genes Dev. 2003;17:603–614. [PMC free article] [PubMed]
22. Hegan DC, et al. Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6. Carcinogenesis. 2006;27:2402–2408. [PMC free article] [PubMed]
23. Shao C, et al. Loss of heterozygosity and point mutation at Aprt locus in T cells and fibroblasts of Pms2−/− mice. Oncogene. 2002;21:2840–2845. [PubMed]
24. Kunkel TA, Pavlov YI, Bebenek K. Functions of human DNA polymerases eta, kappa and iota suggested by their properties, including fidelity with undamaged DNA templates. DNA Repair (Amst) 2003;2:135–149. [PubMed]
25. Kanao R, Hanaoka F, Masutani C. A novel interaction between human DNA polymerase eta and MutLalpha. Biochem Biophys Res Commun. 2009;389:40–45. [PubMed]
26. Edelmann W, et al. Tumorigenesis in Mlh1 and Mlh1/Apc1638N mutant mice. Cancer Res. 1999;59:1301–1307. [PubMed]
27. Lowsky R, et al. Defects of the mismatch repair gene MSH2 are implicated in the development of murine and human lymphoblastic lymphomas and are associated with the aberrant expression of rhombotin-2 (Lmo-2) and Tal-1 (SCL) Blood. 1997;89:2276–2282. [PubMed]
28. Schrader CE, Vardo J, Stavnezer J. Mlh1 can function in antibody class switch recombination independently of Msh2. J Exp Med. 2003;197:1377–1383. [PMC free article] [PubMed]
29. Mayorov VI, Rogozin IB, Adkison LR, Gearhart PJ. DNA polymerase eta contributes to strand bias of mutations of A versus T in immunoglobulin genes. J Immunol. 2005;174:7781–7786. [PubMed]
30. Steele EJ. Mechanism of somatic hypermutation: Critical analysis of strand biased mutation signatures at A:T and G:C base pairs. Mol Immunol. 2009;46:305–320. [PubMed]
31. Gong JG, et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature. 1999;399:806–809. [PubMed]
32. Kadyrov FA, et al. A possible mechanism for exonuclease 1 independent eukaryotic mismatch repair. Proc Natl Acad Sci USA. 2009;106:8495–8500. [PMC free article] [PubMed]
33. Dzantiev L, et al. A defined human system that supports bidirectional mismatch-provoked excision. Mol Cell. 2004;15:31–41. [PubMed]
34. Fang WH, Modrich P. Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction. J Biol Chem. 1993;268:11838–11844. [PubMed]
35. Blackwell LJ, Wang S, Modrich P. DNA chain length dependence of formation and dynamics of hMutSalpha.hMutLalpha.heteroduplex complexes. J Biol Chem. 2001;276:33233–33240. [PubMed]
36. Wong E, et al. Mbd4 inactivation increases C→T transition mutations and promotes gastrointestinal tumor formation. Proc Natl Acad Sci USA. 2002;99:14937–14942. [PMC free article] [PubMed]
37. Dycaico MJ, et al. The use of shuttle vectors for mutation analysis in transgenic mice and rats. Mutat Res. 1994;307:461–478. [PubMed]
38. MacCarthy T, Roa S, Scharff MD, Bergman A. SHMTool: A webserver for comparative analysis of somatic hypermutation datasets. DNA Repair. 2008;8:137–141. [PMC free article] [PubMed]
39. Roa S, et al. Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression. Proc Natl Acad Sci USA. 2008;105:16248–16253. [PMC free article] [PubMed]
40. Kolas NK, et al. Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I. J Cell Biol. 2005;171:447–458. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...