Reconstitution of 5′-directed human mismatch repair in a purified system

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