Xeroderma pigmentosum complementation group A protein (XPA) modulates RPA-DNA interactions via enhanced complex stability and inhibition of strand separation activity.
Replication protein A (RPA) participates in many cellular functions including DNA replication and nucleotide excision repair. A direct interaction between RPA and the xeroderma pigmentosum group A protein (XPA) facilitates the assembly of a preincision complex during the processing of DNA damage by the nucleotide excision repair pathway. We demonstrate here the formation of a ternary RPA, XPA, and duplex cisplatin-damaged DNA complex as is evident by electrophoretic supershift analysis. The RPA-XPA complex displays modest specificity for damaged versus undamaged duplex DNA, and the RPA-XPA complex displays a greater affinity for binding duplex cisplatin-damaged DNA when compared with the RPA or XPA proteins alone, consistent with previous results. Using DNA denaturation assays, we demonstrate that the role of XPA is in the stabilization of the duplex DNA structure via inhibition of the strand separation activity of RPA. Rapid kinetic analysis indicates that the bimolecular k(on) of the RPA-XPA complex is 2.5-fold faster than RPA alone for binding a duplex cisplatin-damaged DNA. The dissociation rate, k(off), of the RPA-XPA complex is slower than that of the RPA protein alone, suggesting that the XPA protein stabilizes the initial binding of RPA to duplex DNA as well as maintaining the integrity of the duplex DNA. Interestingly, XPA has no effect on the k(on) of RPA for a single-stranded 40-mer DNA.
Patrick
Steve M
SM
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Turchi
John J
JJ
D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D051716:Replication Protein A; D013379:Substrate Specificity; D014983:Xeroderma Pigmentosum / Q000235:genetics; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; C497167:RPA1 protein, human; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA; D002945:Cisplatin