title: Reconstitution of 5'-directed human mismatch repair in a purified system.
abstract: 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.
authors.affiliation: Graduate Center for Toxicology and Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA.
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Fenghua
authors.initials: F
authors.lastname: Yuan
authors.forename: Steven R
authors.initials: SR
authors.lastname: Presnell
authors.forename: Keli
authors.initials: K
authors.lastname: Tian
authors.forename: Yin
authors.initials: Y
authors.lastname: Gao
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
authors.forename: Liya
authors.initials: L
authors.lastname: Gu
authors.forename: Guo-Min
authors.initials: GM
authors.lastname: Li
mesh_terms: D024506:5' Flanking Region / Q000502:physiology*; D020137:Base Pair Mismatch; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D024243:HMGB1 Protein / Q000378:metabolism; D006801:Humans; D000070956:MutL Proteins; D009363:Neoplasm Proteins / Q000378:metabolism; D051716:Replication Protein A
chemical_list: D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; C000607892:LIG1 protein, human; C500929:MutLalpha protein, human; D009363:Neoplasm Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; D000070956:MutL Proteins; D011088:DNA Ligases; D045643:DNA Repair Enzymes; D000072481:DNA Ligase ATP