RPA stabilizes the XPA-damaged DNA complex through protein-protein interaction.
The xeroderma pigmentosum group A complementing protein (XPA) and eukaryotic replication protein A (RPA) are among the major damage-recognition proteins involved in the early stage of nucleotide excision repair (NER). XPA and RPA are able to bind damaged DNA independently, although RPA interaction stimulates XPA binding to damaged DNA [Li, L., Lu, X., Peterson, C. A., and Legerski, R. J. (1995) Mol. Cell. Biol. 15, 5396-5402 (1); Stigger, E., Drissi, R., and Lee, S.-H. (1998) J. Biol. Chem. 273, 9337-9343 (2)]. In this study, we used surface plasmon resonance (SPR) analysis to investigate the interaction of XPA and RPA with two major types of UV-damaged DNA: the (6-4) photoproduct and the cis-syn cyclobutane dimer of thymidine. Both XPA and RPA preferentially bind to (6-4) photoproduct-containing duplex DNA over cis-syn cyclobutane dimer-containing DNA. The binding of XPA to (6-4) photoproduct was weak (K(D) = 2.13 x 10(-)(8) M), whereas RPA showed a very stable interaction with (6-4) photoproduct (K(D) = 2. 02 x 10(-)(10) M). When XPA and RPA were incubated together, the stability of the XPA-damaged DNA interaction was significantly enhanced by wild-type RPA. On the other hand, mutant RPA (RPA:p34Delta33C) defective in its interaction with XPA failed to stabilize XPA-damaged DNA complex. Taken together, our results suggest that a role for RPA in UV-damage recognition is to stabilize XPA-damaged DNA complex through protein-protein interaction.
Wang
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Department of Biochemistry & Molecular Biology, Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis 46202, USA.
Mahrenholz
A
A
Lee
S H
SH
D001483:Base Sequence; D019921:Biotinylation; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011740:Pyrimidine Dimers; D051716:Replication Protein A; D020349:Surface Plasmon Resonance; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; C497167:RPA1 protein, human; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein