Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends
Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends
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DOI：
10.1093/nar/gkh761
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发表时间：
2004-08-01
影响因子：
14.9
通讯作者：
Lees-Miller, SP
中科院分区：
文献类型：
--
作者：
Block, WD;Yu, YP;Lees-Miller, SP
非同源末端连接（NHEJ）是电离辐射（IR）诱导的DNA双链断裂（DSB）修复的主要途径之一。NHEJ所需的蛋白质包括DNA依赖性蛋白激酶（DNA-PKcs）的催化亚基、Ku、XRCC 4和DNA连接酶IV。目前的模型预测，DNA-PKcs，Ku，XRCC 4和DNA连接酶IV组装在DSB和蛋白激酶活性的DNA-PKcs是必不可少的NHEJ介导的修复DSB在体内。我们以前确定了DNA-PKcs的氨基酸2609和2647之间的自磷酸化位点簇。表达DNA-PKcs的细胞，其中这些自磷酸化位点已突变为丙氨酸，是高度放射敏感性的，并且在染色体外测定的情况下，其修复DSB的能力有缺陷。在这里，我们表明，细胞表达DNA-PKcs突变的自动磷酸化位点也有缺陷的修复IR诱导的DSB的背景下，染色质。纯化的DNA-PKcs蛋白含有丝氨酸/苏氨酸丙氨酸或天冬氨酸突变，在这一簇的自磷酸化位点的野生型（野生型）蛋白质的蛋白激酶活性是无法区分的。然而，突变体DNA-PKcs蛋白相对于野生型DNA-PKcs在其支持T4 DNA连接酶介导的DNA末端分子间连接的能力方面是有缺陷的。我们建议，DNA-PKcs在这一簇网站的自磷酸化是重要的DNA末端连接之前，在DNA末端的DNA-PK复合物的重塑。
Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated auto phosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.