Summary DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 tri-methyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.