Jones C J, Wood R D
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, U.K.
Biochemistry. 1993 Nov 16;32(45):12096-104. doi: 10.1021/bi00096a021.
The xeroderma pigmentosum group A complementing protein (XPAC) is involved in an early step of nucleotide excision repair, the main process that removes UV damage and many chemical lesions from DNA. To explore the properties and function of XPAC, recombinant protein encoded by the human XPAC cDNA was expressed with an N-terminal polyhistidine tag in Escherichia coli and purified to homogeneity. The soluble fusion protein could correct the repair defect in vitro of XP-A cell extracts. XPAC protein bound to DNA with a preference for UV-irradiated over nonirradiated DNA, as determined by a gel electrophoresis mobility shift assay with a 258 base pair DNA fragment (the association constant was approximately 3 x 10(6) M-1 for the fragment irradiated with 6 kJ/m2 UV light). Removal of cyclobutane pyrimidine dimers from UV-irradiated DNA by enzymatic photoreactivation did not significantly reduce binding of XPAC to the irradiated fragment, indicating that binding was mostly due to (6-4) photoproducts, with a preference for a (6-4) photoproduct over an undamaged base pair up to 300-fold. Undamaged single-stranded DNA competed about 4-fold more effectively than undamaged double-stranded DNA for binding of XPAC to a UV-irradiated fragment. In addition, XPAC bound to DNA treated with the chemotherapeutic agent cis-diamminedichloroplatinum(II). The results suggest that XPAC functions as a key component in recognition of DNA damage during repair.
着色性干皮病A组互补蛋白（XPAC）参与核苷酸切除修复的早期步骤，核苷酸切除修复是从DNA中去除紫外线损伤和许多化学损伤的主要过程。为了探究XPAC的特性和功能，由人XPAC cDNA编码的重组蛋白在大肠杆菌中表达，并带有N端多组氨酸标签，然后纯化至同质。可溶性融合蛋白可在体外纠正XP - A细胞提取物的修复缺陷。通过使用258个碱基对的DNA片段进行凝胶电泳迁移率变动分析确定，XPAC蛋白与DNA结合，相较于未受紫外线照射的DNA，它更倾向于与紫外线照射过的DNA结合（对于用6 kJ/m2紫外线照射的片段，结合常数约为3×10(6) M-1）。通过酶促光复活从紫外线照射的DNA中去除环丁烷嘧啶二聚体，并不会显著降低XPAC与照射片段的结合，这表明结合主要是由于（6 - 4）光产物，对（6 - 4）光产物的偏好比对未受损碱基对高300倍。未受损的单链DNA在竞争XPAC与紫外线照射片段的结合时，比未受损的双链DNA有效约4倍。此外，XPAC与用化疗药物顺二氯二氨铂（II）处理的DNA结合。结果表明，XPAC在修复过程中作为识别DNA损伤的关键成分发挥作用。
