Partial reconstitution of human interstrand cross-link repair in vitro: characterization of the roles of RPA and PCNA.
The repair of DNA interstrand cross-links (ICLs) remains largely ill-defined in higher eukaryotic cells. Previously, we have developed assays that can be used to monitor the early stages of processing of ICLs in vitro. Here, we have used P11 phosphocellulose chromatography to fractionate HeLa nuclear extracts and have subsequently reconstituted these assays with the resulting fractions. RPA and PCNA were found in a single fraction, and were the only factors in this fraction required for the reconstitution of these assays. The roles of RPA and PCNA in the formation of incisions at ICLs and in the subsequent DNA synthesis step were assessed. RPA was found to be essential for both stages of ICL processing indicating that it is required for lesion recognition and/or for the subsequent endonucleolytic processing. PCNA is required for the DNA synthesis stage and although it is not critical for the incision stage of the reaction it does enhance this step presumably by a stimulation of lesion recognition by MutSbeta. These findings define novel roles for RPA and PCNA in the processing of ICLs in mammalian cells.
Zhang
Nianxiang
N
Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Lu
Xiaoyan
X
Legerski
Randy J
RJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins; D020137:Base Pair Mismatch; D002467:Cell Nucleus / Q000378:metabolism; D002845:Chromatography; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004720:Endonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D051719:MutS DNA Mismatch-Binding Protein; D010957:Plasmids / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051716:Replication Protein A; D013347:Subcellular Fractions / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli