Human Mismatch Repair RECONSTITUTION OF A NICK-DIRECTED BIDIRECTIONAL REACTION

摘要: Bidirectional mismatch repair directed by a strand break located 3′ or 5′ to the mispair has been reconstituted using seven purified human activities: MutSα, MutLα, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), factor C (RFC) and DNA polymerase δ. In addition δ, PCNA, RFC, RPA, 5′-directed depends on MutSα whereas 3′-directed correction also requires MutLα. The reaction displays specificity for an effect that presumably reflects interactions with other activities. Because previous studies have suggested potential involvement of editing function replicative in mismatch-provoked excision, we evaluated possible participation δ excision step repair. RFC PCNA dramatically activate δ-mediated hydrolysis primer-template. Nevertheless, contribution is very limited, both system HeLa extracts, as judged vitro assay nicked circular heteroplex DNAs. Thus, containing are reduced 10-fold upon omission EXOI substitution catalytically dead form exonuclease. Furthermore, aphidicolin inhibits 3′- extracts only 20–30%. Although this modest inhibition could be because nonspecific effects, it may indicate limited dependence bidirectional aphidicolin-sensitive polymerase.