AN INTERACTION BETWEEN THE MAMMALIAN DNA-REPAIR PROTEIN XRCC1 AND DNA LIGASE-III
AN INTERACTION BETWEEN THE MAMMALIAN DNA-REPAIR PROTEIN XRCC1 AND DNA LIGASE-III
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DOI：
10.1128/mcb.14.1.68
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发表时间：
1994-01-01
影响因子：
5.3
通讯作者：
THOMPSON, LH
中科院分区：
文献类型：
--
作者：
CALDECOTT, KW;MCKEOWN, CK;THOMPSON, LH
XRCC1是一种完全纠正中国仓鼠卵巢DNA修复突变体EM9的人类基因，编码一种蛋白质，参与烷化剂或电离辐射处理后出现的DNA单链断裂的重新连接。在这项研究中，一个cDNA minigene编码寡组氨酸标记的XRCC 1的构建，以促进亲和纯化的重组蛋白。该构建体命名为pcD2EHX，完全纠正了高姐妹染色单体交换的EM9表型，表明组氨酸标签对XRCC1活性无害。亲和层析的提取物从EM 9细胞转染pcD2EHX导致共纯化组氨酸标记的XRCC 1和DNA连接酶III的活性。无论是XRCC1或DNA连接酶III的活性进行纯化，在亲和层析提取物从EM9细胞转染pcD2EX，cDNA小基因编码未标记的XRCC1，或提取物从野生型AA8或未转染的EM9细胞。DNA连接酶III活性与组氨酸标记的XRCC 1的共纯化表明这两种蛋白质作为复合物存在于细胞中。此外，DNA连接酶III活性在EM9细胞中的水平低于AA8细胞，并且在用pcD2EHX或pcD2EX转染的EM9细胞中恢复到正常水平。这些研究结果表明，XRCC1是所需的正常水平的DNA连接酶III活性，他们牵连的主要作用，这种DNA连接酶在哺乳动物细胞中的DNA碱基切除修复。
XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.