Functional analysis of human replication protein A in nucleotide excision repair.
Human replication protein A (RPA) is a three-subunit protein complex (70-, 34-, and 11-kDa subunits) involved in DNA replication, repair, and recombination. Both the 70- (p70) and 34-kDa (p34) subunits interact with Xeroderma pigmentosum group A complementing protein (XPA), a key protein involved in nucleotide excision repair. Our deletion analysis indicated that no particular domain(s) of RPA p70 was essential for its interaction with XPA, whereas 33 amino acids from the C terminus of p34 (p34Delta33C) were necessary for the XPA interaction. Furthermore, mutant RPA lacking the p34 C terminus failed to interact with XPA, suggesting that p34, not p70, is primarily responsible for the interaction of RPA with XPA. RPA stimulated the interaction of XPA with UV-damaged DNA through an RPA-XPA complex on damaged DNA sites because (i) the RPA mutant lacking the C terminus of p34 failed to stimulate an XPA-DNA interaction, and (ii) the ssDNA binding domain of RPA (amino acids 296-458) was necessary for the stimulation of the XPA-DNA interaction. Two separate domains of p70, a single-stranded DNA binding domain and a zinc-finger domain, were necessary for RPA function in nucleotide excision repair. The mutant RPA (RPA:p34Delta33C), which lacks its stimulatory effect on the XPA-DNA interaction, also poorly supported nucleotide excision repair, suggesting that the XPA-RPA interaction on damaged DNA is necessary for DNA repair activity.
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Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Drissi
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Lee
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D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000528:radiation effects; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA