Robins P, Jones C J, Biggerstaff M, Lindahl T, Wood R D
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
EMBO J. 1991 Dec;10(12):3913-21. doi: 10.1002/j.1460-2075.1991.tb04961.x.
Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins.
着色性干皮病（XP）的互补组A代表了这种易患癌症的疾病中最常见和最严重的形式之一。由于DNA切除修复存在明显缺陷，XP - A患者的细胞对紫外线和许多化学试剂的毒性和诱变作用高度敏感。我们在此报告通过对一种修复缺陷型人类XP - A细胞系的细胞提取物进行互补作用来分离XP - A DNA修复蛋白。从小牛胸腺纯化的XP - A蛋白在变性凝胶电泳上迁移时呈现为40和42千道尔顿的双峰。XP - A蛋白优先结合紫外线照射的DNA，对受损核苷酸与未受损核苷酸的偏好约为10³。这强烈表明XP - A蛋白在识别和切割DNA损伤中起直接作用。我们进一步表明，这种蛋白对应于最近分离的一个基因所编码的产物，该基因可恢复XP - A细胞的切除修复。因此，细胞提取物对质粒DNA的切除修复与细胞中的基因组修复非常相似，足以准确揭示一种遗传性疾病中的修复缺陷。这里描述的一般方法可扩展到鉴定和分离其他人类DNA修复蛋白。
