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 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.
Zhang
Yanbin
Y
Graduate Center for Toxicology and Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA.
Yuan
Fenghua
F
Presnell
Steven R
SR
Tian
Keli
K
Gao
Yin
Y
Tomkinson
Alan E
AE
Gu
Liya
L
Li
Guo-Min
GM
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
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