Reaction mechanism of human DNA repair excision nuclease
Reaction mechanism of human DNA repair excision nuclease
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DOI：
10.1074/jbc.271.14.8285
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发表时间：
1996-04-05
影响因子：
4.8
通讯作者：
Sancar, A
中科院分区：
文献类型：
--
作者：
Mu, D;Hsu, DS;Sancar, A
核苷酸切除修复包括通过在损伤两侧上双切割受损链从DNA中去除受损的核苷酸，然后填充所得缺口并连接。在人类中，双切口步骤需要14-16个多肽。我们已经将所需的蛋白质纯化至同质，并在确定的酶/底物系统中重建双切割活性（切除核酸酶）。该系统是高效的，在最佳条件下去除>30%的胸腺嘧啶二聚体。构成切除核酸酶的所有六个级分对于胸腺嘧啶二聚体底物的双切割是必需的。然而，当使用胆固醇取代的寡核苷酸作为底物时，在不存在XPC-HHR 23 B复合物的情况下发生切除，这让人想起XP-C突变细胞系中的转录偶联修复。两个切口都需要复制蛋白A。XPG亚基对于切口前复合体的形成是必不可少的，但是修复复合体可以在XPF-ERCC 1不存在的情况下组装并产生正常水平的3 '-切口。动力学实验表明，3 '切口先于5'切口。与动力学数据一致，在复溶系统中从未观察到解偶联的5 '-切口。两种形式的TFIIH用于重组反应，一种含有CDK 7-细胞周期蛋白H对，另一种缺乏CDK 7-细胞周期蛋白H对。切除的寡聚体从核蛋白复合物中的缺口DNA解离。总的来说，这些结果提供了一个详细的说明过程中发生的损伤去除人切除核酸酶的反应。
Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14-16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3'-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3'-incision precedes the 5'-incision. Consistent with the kinetic data, uncoupled 5'-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease.