The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites
The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites
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DOI：
10.1074/jbc.m112.369975
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发表时间：
2012-11-09
影响因子：
4.8
通讯作者：
Tomkinson, Alan E.
中科院分区：
文献类型：
--
作者：
Della-Maria, Julie;Hegde, Muralidhar L.;Tomkinson, Alan E.
XRCC 1在DNA碱基损伤和单链断裂的修复中起着关键作用。虽然它没有已知的酶活性，但XRCC 1与多种DNA修复蛋白相互作用，并且是不同DNA修复蛋白复合物的亚基。在这里，我们使用酵母双杂交遗传分析来鉴定XRCC 1的突变版本，这些突变版本在与单一蛋白质伴侣的相互作用中有选择性缺陷。一个XRCC 1突变体A482 T在与多核苷酸激酶磷酸酶（PNKP）结合方面有缺陷，不仅保留了与结合XRCC 1不同区域的伴侣蛋白相互作用的能力，而且还保留了与结合位点与PNKP重叠的aprataxin和aprataxin样因子相互作用的能力。PNKP和XRCC 1之间相互作用的破坏并不影响它们在局部DNA损伤位点的初始募集，但显著降低了它们在那里的保留。此外，PNKP和DNA连接酶III α-XRCC 1复合物之间的相互作用显著增加了重建修复反应的效率，并且是补充xrcc 1突变细胞对DNA烷基化剂的DNA损伤敏感性所必需的。总之，我们的研究结果揭示了PNKP和XRCC 1之间的相互作用在DNA损伤位点和DNA烷基化损伤修复中保留XRCC 1的新作用。
XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase III alpha-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.