Reconstitution of 5-Directed Human Mismatch Repair in a Purified System

This paper reports reconstitution of 5ʹ-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 MutS (alpha), which efficiently corrects both types of heteroduplexes. MutL(alpha) reduces the processivity of EXO1 and terminates EXO1catalyzed 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.