Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.
Bidirectional mismatch repair directed by a strand break located 3' or 5' to the mispair has been reconstituted using seven purified human activities: MutSalpha, MutLalpha, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase delta. In addition to DNA polymerase delta, PCNA, RFC, and RPA, 5'-directed repair depends on MutSalpha and EXOI, whereas 3'-directed mismatch correction also requires MutLalpha. The repair reaction displays specificity for DNA polymerase delta, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase delta in the excision step of repair. RFC and PCNA dramatically activate polymerase delta-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase delta are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3'- and 5'-directed excision in HeLa nuclear extracts by only 20-30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase.
Constantin
Nicoleta
N
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Dzantiev
Leonid
L
Kadyrov
Farid A
FA
Modrich
Paul
P
D016590:Aphidicolin / Q000494:pharmacology; D020137:Base Pair Mismatch; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004258:DNA Polymerase III / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry; D051716:Replication Protein A / Q000737:chemistry; D051818:Replication Protein C / Q000737:chemistry
D017931:DNA Primers; C500929:MutLalpha protein, human; D009363:Neoplasm Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D016590:Aphidicolin; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; D005092:Exonucleases; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D051818:Replication Protein C; D045643:DNA Repair Enzymes