Human polynucleotide kinase participates in repair of DNA double-strand breaks by nonhomologous end joining but not homologous recombination
Human polynucleotide kinase participates in repair of DNA double-strand breaks by nonhomologous end joining but not homologous recombination
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DOI：
10.1158/0008-5472.can-07-0480
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发表时间：
2007-07-15
期刊：
影响因子：
11.2
通讯作者：
Weinfeld, Michael
中科院分区：
文献类型：
--
作者：
Karimi-Busheri, Feridoun;Rasouli-Nia, Aghdass;Weinfeld, Michael
人多核苷酸激酶（hPNK）是一种具有5 '-DNA激酶活性和3'-磷酸酶活性的双功能酶。基于细胞提取物和纯化蛋白的研究表明，hPNK可以作用于单链断裂和双链断裂（DSB），以将末端恢复为DNA修复聚合酶进一步作用所需的化学形式。和连接酶（即，5 ′-磷酸和3 ′-羟基末端）。这些研究表明，hPNK可以与XRCC 4结合。因此，hPNK被认为是DSB修复的非同源末端连接（NHEJ）途径的参与者。我们试图使用遗传方法来确认hPNK在细胞环境中NHEJ中的作用。在NHEJ阳性的M059 K胶质母细胞瘤细胞和由于缺乏DNA-PK催化亚基（DNA-PKcs）而NHEJ缺陷的M059 J细胞中，hPNK通过RNA干扰表达稳定下调。尽管hPNK的耗尽显著地使M059 K细胞对电离辐射敏感，但没有赋予M059 J细胞额外的敏感性，这清楚地表明hPNK在与DNA-PKcs相同的DNA修复途径中起作用。另一方面，耗尽hPNK并没有增加姐妹染色单体交换的水平，表明hPNK不参与同源重组DSB修复途径。我们还提供了证据表明，hPNK在修复喜树碱诱导的拓扑异构酶1“死端”复合物中的作用不依赖于DNA-PKcs，并且hPNK不参与核苷酸切除修复途径。
Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5'-DNA kinase activity and a 3'-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases. and ligases (i.e., 5'-phosphate and 3'-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4. and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells,which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also pro-side evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 "dead-end" complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway.