Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase
Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase
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DOI：
10.1093/nar/gkg775
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发表时间：
2003-10-15
影响因子：
14.9
通讯作者：
Dynan, WS
中科院分区：
文献类型：
--
作者：
Li, SY;Takeda, Y;Dynan, WS
非同源末端连接途径使用预先存在的蛋白质来修复电离辐射诱导的DNA双链断裂。在这里，我们描述了使用新开发的工具在活细胞中操纵这一途径。我们产生了一个单链抗体可变片段（scFv），结合到DNA依赖性蛋白激酶催化亚基（DNA-PKcs），在途径中的关键酶。与现有的药理学抑制剂相比，scFv结合激酶催化结构域之外的新定义的调节位点。尽管scFv仅适度抑制激酶活性，但其在无细胞系统中完全阻断DNA末端连接。scFv的显微注射使人细胞对辐射敏感，如在1.5戈伊的亚致死剂量下通过集落形成效率和细胞凋亡诱导的降低所测量的。scFv在组蛋白γ-H2 AX焦点形成之后但在γ-H2 AX去磷酸化之前的步骤中原位阻断非同源末端连接。阻断发生在暴露于低至0.1戈伊的细胞中，表明DNA-PKcs即使在低辐射剂量下也是双链断裂修复所必需的。在活细胞中原位修改辐射反应的能力提供了生物化学、遗传学和细胞学方法之间的联系，以研究双链断裂修复中间体。
The non-homologous end joining pathway uses pre-existing proteins to repair DNA double-strand breaks induced by ionizing radiation. Here we describe manipulation of this pathway in living cells using a newly developed tool. We generated a single chain antibody variable fragment (scFv) that binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in the pathway. In contrast to existing pharmacologic inhibitors, the scFv binds a newly defined regulatory site outside the kinase catalytic domain. Although the scFv inhibits kinase activity only modestly, it completely blocks DNA end joining in a cell-free system. Microinjection of the scFv sensitizes human cells to radiation, as measured by a reduction in efficiency of colony formation and induction of apoptosis at an otherwise sublethal dose of 1.5 Gy. The scFv blocks non-homologous end joining in situ at a step subsequent to histone gamma-H2AX focus formation but preceding gamma-H2AX dephosphorylation. Blockage occurs in cells exposed to as little as 0.1 Gy, indicating that DNA-PKcs is essential for double-strand break repair even at low radiation doses. The ability to modify the radiation response in situ in living cells provides a link between biochemical, genetic and cytologic approaches to the study of double-strand break repair intermediates.