用纯化的蛋白质成分重建的哺乳动物DNA核苷酸切除修复。
Mammalian DNA nucleotide excision repair reconstituted with purified protein components.
作者信息
Aboussekhra A, Biggerstaff M, Shivji M K, Vilpo J A, Moncollin V, Podust V N, Protić M, Hübscher U, Egly J M, Wood R D
机构信息
Imperial Cancer Research Fund, Clare Hall Laboratoires, South Mimms, Herts, England.
出版信息
Cell. 1995 Mar 24;80(6):859-68. doi: 10.1016/0092-8674(95)90289-9.
Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.
核苷酸切除修复是人类细胞从DNA中去除紫外线损伤的主要方式。对人类细胞提取物进行分级分离以定位活性成分，包括着色性干皮病（XP）和ERCC因子。然后用纯化的蛋白质RPA、XPA、TFIIH（包含XPB和XPD）、XPC、UV-DDB、XPG、部分纯化的ERCC1/XPF复合物以及一种名为IF7的因子重建切口反应。UV-DDB（与XPE蛋白相关）刺激修复，但并非必不可少。ERCC1和XPF校正活性与一种110 kDa的ERCC1结合多肽共纯化，该多肽在XP-F细胞提取物中不存在。通过将这些因子与DNA聚合酶ε、RFC、PCNA和DNA连接酶I相结合实现了完全修复合成。重建的核心反应需要约30种多肽。
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