title: Photo-cross-linking of XPC-Rad23B to cisplatin-damaged DNA reveals contacts with both strands of the DNA duplex and spans the DNA adduct.
abstract: Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The xeroderma pigmentosum group C (XPC)-Rad23B complex is involved in the recognition of these bulky DNA adducts and initiates the global genomic nucleotide excision repair pathway (GG-NER). Photo-cross-linking experiments revealed that the human XPC-Rad23B complex makes direct contact with both the cisplatin-damaged DNA strand and the complementary undamaged strand of a duplex DNA substrate. Coupling photo-cross-linking with denaturation and immunoprecipitation of protein-DNA complexes, we identified the XPC subunit in complex with damaged DNA. While the interaction of the XPC subunit with DNA was direct, studies revealed that although Rad23B was found in complex with DNA, the Rad23B-DNA interaction was largely indirect via its interaction with XPC. Using site specific cross-linking, we determined that the XPC-Rad23B complex is preferentially cross-linked to the damaged DNA when the photoreactive FAP-dCMP (exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine 5'-monophosphate) analogue is located to the 5' side of the cisplatin-DNA adduct. When the FAP-dCMP analogue is located to the 3' side of the adduct, no difference in binding was detected between undamaged and damaged DNA. Collectively, these data suggest a model in which XPC-DNA interactions drive the damage recognition process contacting both the damaged and undamaged DNA strand. Preferential cross-linking 5' of the cisplatin-damaged site suggests that the XPC-Rad23B complex displays orientation specific binding to eventually impart directionality to the downstream binding and incision events relative to the site of DNA damage.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Neher
authors.forename: Nadejda I
authors.initials: NI
authors.lastname: Rechkunova
authors.forename: Olga I
authors.initials: OI
authors.lastname: Lavrik
authors.forename: John J
authors.initials: JJ
authors.lastname: Turchi
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002945:Cisplatin / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003843:Deoxycytidine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D014983:Xeroderma Pigmentosum / Q000378:metabolism
chemical_list: D001386:Azides; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; C547827:N-(2-(N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl)aminoethyl)-2'-deoxycytidine 5'-monophosphate; C057270:cisplatin-DNA adduct; D003843:Deoxycytidine Monophosphate; D004247:DNA; D002945:Cisplatin