Reconstitution of DNA base excision-repair with purified human proteins: Interaction between DNA polymerase beta and the XRCC1 protein
Reconstitution of DNA base excision-repair with purified human proteins: Interaction between DNA polymerase beta and the XRCC1 protein
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DOI：
10.1002/j.1460-2075.1996.tb01056.x
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发表时间：
1996-12-02
期刊：
影响因子：
11.4
通讯作者：
Lindahl, T
中科院分区：
文献类型：
--
作者：
Kubota, Y;Nash, RA;Lindahl, T
DNA中尿嘧啶-鸟嘌呤碱基对的修复已经用重组人蛋白尿嘧啶-DNA糖基化酶、脱嘌呤/脱嘧啶核酸内切酶、DNA聚合酶β和DNA连接酶III重建。已知结合DNA连接酶III的XRCC 1蛋白不是反应绝对需要的，但抑制DNA聚合酶β的链置换，允许在填充单核苷酸补丁后更有效的连接，我们用远Western印迹、亲和沉淀和酵母双杂交分析表明，XRCC 1与DNA聚合酶β直接相互作用。此外，在凝胶阻滞试验中，DNA聚合酶β与含有切割的脱碱基位点的双链寡核苷酸之间形成的复合物被XRCC 1超移，与DNA聚合酶β相互作用的区域位于XRCC 1蛋白N-末端一半的残基84-183内，而XRCC 1的C-末端区域参与结合DNA连接酶III。这些数据表明，XRCC 1，它没有已知的催化活性，可能作为一个支架蛋白在碱基切除修复，DNA链置换和过度的缺口填充在DNA修复过程中观察到的XRCC 1缺陷突变细胞系的无细胞提取物，与重建系统的结果一致。
Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III, The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch, We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses, In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay, The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair, DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.