Photocrosslinking of XPC-Rad23B to cisplatin-damaged DNA reveals contacts with both strands of the DNA duplex and spans the DNA adduct

摘要

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). Photocrosslinking 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 photocrosslinking with denaturation and immunoprecipitation of protein-DNA complexes, the XPC subunit was identified 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 crosslinking we determined that XPC-Rad23B is preferentially crosslinked to the damaged DNA when the photoreactive FAP-dCMP (exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoethyl}-2′-deoxycytidine-5′-monophosphate) analogue is located 5′ of the cisplatin-DNA adduct. When the FAP-dCMP analogue is located 3′ of the adduct, no difference in binding was detected between undamaged and damaged DNA. Collectively, these data suggest a model whereby XPC-DNA interactions drive the damage recognition process contacting both the damaged and undamaged DNA strand. Preferential crosslinking 5′ of the cisplatin-damaged site suggests that XPC-Rad23B displays orientation specific binding to eventually impart directionality to the downstream binding and incision events relative to the site of DNA damage.