Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Biochemistry. 2010 Feb 2;49(4):669-78. doi: 10.1021/bi901575h.
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.
核苷酸切除修复（NER）是用于修复大体积 DNA 加合物的主要途径，例如由紫外线照射和化疗药物顺铂引起的加合物。着色性干皮病组 C（XPC）-Rad23B 复合物参与这些大体积 DNA 加合物的识别，并启动全基因组核苷酸切除修复途径（GG-NER）。光交联实验表明，人 XPC-Rad23B 复合物与顺铂损伤的 DNA 链以及双链 DNA 底物的互补未损伤链直接接触。通过光交联与蛋白质-DNA 复合物的变性和免疫沉淀相结合，我们鉴定了与损伤 DNA 结合的 XPC 亚基。虽然 XPC 亚基与 DNA 的相互作用是直接的，但研究表明，尽管 Rad23B 与 DNA 结合，但 Rad23B-DNA 相互作用主要是通过与 XPC 的相互作用间接进行的。使用定点交联，我们确定当光反应性 FAP-dCMP（外切-N-（2-[[N-（4-叠氮基-2,5-二氟-3-氯吡啶-6-基）-3-氨基丙酰基]氨基]乙基]-2'-脱氧胞苷 5'-单磷酸）类似物位于顺铂-DNA 加合物的 5'侧时，XPC-Rad23B 复合物优先交联到损伤的 DNA。当 FAP-dCMP 类似物位于加合物的 3'侧时，未损伤和损伤 DNA 之间的结合没有差异。总的来说，这些数据表明了一种模型，其中 XPC-DNA 相互作用驱动损伤识别过程，同时接触损伤和未损伤的 DNA 链。在顺铂损伤部位的 5'处优先交联表明，XPC-Rad23B 复合物对最终赋予相对于 DNA 损伤位点的下游结合和切口事件的方向性表现出定向特异性结合。
