Binding of XPA and RPA to damaged DNA investigated by fluorescence anisotropy.
The proteins XPA and RPA are assumed to be involved in primary damage recognition of global genome nucleotide excision repair. XPA as well as RPA have been each reported to specifically bind DNA lesions, and ternary complex formation with damaged DNA has also been shown. We employed fluorescence anisotropy measurements to study the DNA-binding properties of XPA and RPA under true equilibrium conditions using damaged DNA probes carrying a terminal fluorescein modification as a reporter. XPA binds with low affinity and in a strongly salt-dependent manner to DNA containing a 1,3-d(GTG) intrastrand adduct of the anticancer drug cisplatin or a 6-nt mismatch (K(D) = 400 nM) with 3-fold preference for damaged vs undamaged DNA. At near physiological salt conditions binding is very weak (K(D) > 2 microM). RPA binds to damaged DNA probes with dissociation constants in the range of 20 nM and a nearly 15-fold preference over undamaged DNA. The presence of a cisplatin modification weakens the affinity of RPA for single-stranded DNA by more than 1 order of magnitude indicating that binding to the lesion itself is not a driving force in damage recognition. Our fluorescence anisotropy assays also show that the presence of XPA does not enhance the affinity of RPA for damaged DNA although both proteins interact. In contrast, cooperative binding of XPA and RPA is observed in EMSA. Our results point to a damage-sensing function of the XPA-RPA complex with RPA mediating the important DNA contacts.
Lehrstuhl für Biochemie, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany.
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D020137:Base Pair Mismatch; D002945:Cisplatin / Q000378:metabolism / Q000494:pharmacology; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D051716:Replication Protein A; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein
D018736:DNA Adducts; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C497167:RPA1 protein, human; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C057270:cisplatin-DNA adduct; D002945:Cisplatin