Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutations at these sites on DNA end joining in a cell-free system
Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutations at these sites on DNA end joining in a cell-free system
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DOI：
10.1016/j.dnarep.2003.11.005
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发表时间：
2004-03-04
期刊：
影响因子：
3.8
通讯作者：
Dynan, WS
中科院分区：
文献类型：
--
作者：
Lee, KJ;Jovanovic, M;Dynan, WS
非同源末端连接（NHEJ）是哺乳动物细胞DNA双链断裂修复的主要机制。NHEJ需要至少三种蛋白质组分：DNA依赖性蛋白激酶催化亚基（DNA-PKcs）、Ku蛋白和DNA连接酶IV/XRCC 4（DNL IV/XRCC 4）复合物。虽然DNA-PKcs磷酸化自身和这些其他蛋白质中的几个位点，但在单个位点磷酸化的意义尚不清楚。在这里，我们调查的影响DNA-PKcs介导的磷酸化在两个网站在XRCC 4。一个是先前描述的丝氨酸260位点;另一个是新绘制的丝氨酸318位点。在这些位点携带突变的XRCC 4与DNL IV共表达，纯化所得复合物，并在由重组和纯化的蛋白质重建的无细胞末端连接系统中测试活性。丙氨酸取代丝氨酸260或318，防止在这些位置的磷酸化，或天冬氨酸取代丝氨酸260，模拟组成性磷酸化，对整体末端连接活性没有显着影响。在使用的检测系统中，DNA-PKcs并不是必需的，但当存在时，会阻止反应，直到磷酸化发生，实际上建立了反应检查点。丝氨酸260和318的突变不影响检查点的建立或释放。结果表明，DNA-PKcs介导的XRCC 4丝氨酸260和丝氨酸318的磷酸化在测试条件下不直接控制末端连接。(C)2003 Elsevier B. V.保留所有权利。
Nonhomologous end joining (NHEJ)is the principal mechanism for repairing DNA double-strand breaks in mammalian cells. NHEJ requires at least three protein components: the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku protein, and the DNA ligase IV/XRCC4 (DNL IV/XRCC4) complex. Although DNA-PKcs phosphorylates several sites within itself and these other proteins, the significance of phosphorylation at individual sites is not yet understood. Here we investigate the effects of DNA-PKcs-mediated phosphorylation at two sites in XRCC4. One is a previously described site at serine 260; the other is a newly mapped site at serine 318. XRCC4 bearing mutations at these sites was co-expressed with DNL IV, the resulting complexes were purified, and activity was tested in a cell-free end-joining system reconstituted from recombinant and purified proteins. Substitution of alanine for serine 260 or 318, which prevents phosphorylation at these positions, or aspartate for serine 260, which mimics constitutive phosphorylation, had no significant effect on overall end-joining activity. In the assay system used, DNA-PKcs is not essential, but when present, arrests the reaction until phosphorylation occurs, in effect establishing a reaction checkpoint. Mutations at serines 260 and 318 did not affect establishment or release from the checkpoint. Results demonstrate that DNA-PKcs-mediated phosphorylation of XRCC4 serine 260 and serine 318 does not directly control end-joining under the conditions tested. (C) 2003 Elsevier B.V. All rights reserved.