The DNA mismatch repair (MMR) system plays an important role in maintaining genomic stability and defects in this system are the pathological basis of hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers with microsatellite instability. However, the molecular mechanism of human MMR is not fully understood. We report here the reconstitution of 5’ nick-directed MMR reaction using purified human proteins. The reconstituted system includes MutSα (MSH2/MSH6) or MutSβ (MSH2/MSH3), MutLα (MLH1/PMS2), RPA, EXO1, HMGB1, PCNA, RFC, DNA polymerase δ, and DNA ligase I. In this system, MutSβ plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSα, which efficiently corrects both types of heteroduplexes. MutLα reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLα, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSα-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role by facilitating MutLα-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSα-MutLα complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision. Given the fact that at least four exonucleases are involved in E. coli mismatch excision, we hypothesize that multiple exonucleases are involved in human mismatch repair. We have indeed identified a novel 5’-3’ mismatch excision activity. Unlike EXO1, the 5’-3’ excision activity is dependent on PCNA for its function. Extensive chromatography revealed that this excision activity contains an exonuclease and a nuclease-stimulatory factor, both of which are indispensable for efficient 5’ nick-directed repair. The stimulating activity was purified to near homogeneity, and appears to co-purify a heterotrimer consisting of 70-kDa, 36-kDa, and 30-kDa polypeptides. We are in the process of revealing the identity of the stimulating factor by mass spectrometry, and purifying and identifying the nuclease activity.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]