Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis.
Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis.
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DOI：
10.1093/nar/gkr257
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发表时间：
2011-08
影响因子：
14.9
通讯作者：
Povirk LF
中科院分区：
文献类型：
--
作者：
Mohapatra S;Kawahara M;Khan IS;Yannone SM;Povirk LF
Artemis缺陷与缺乏V（D）J重组、对辐射和拟辐射药物的敏感性以及未能修复DNA双链断裂（DSB）的子集有关。Artemis具有修剪DSB的5′-和3′-末端的内切核酸酶活性。为了检查Artemis对末端阻断DSB的核酸内切修剪是否是生物学相关功能，Artemis缺陷型成纤维细胞与野生型Artemis或核酸内切酶缺陷型D165 N突变体稳定互补。野生型Artemis完全恢复了对γ射线、博来霉素和新制癌素的抗性，并且通过脉冲场凝胶电泳和修复焦点分辨率测量，还恢复了G 0/G1期的DSB修复能力。相比之下，表达D165 N突变体的细胞，即使在非常高的水平下，仍然与亲本细胞一样具有化学/放射敏感性和修复缺陷，如持久的γ-H2 AX、53 BP 1和Mre 11灶所证明的，这些灶的大小缓慢增加，最终与早幼粒细胞白血病蛋白核体并置。在正常成纤维细胞中，野生型Artemis的过度表达增加了辐射抗性，而D165 N的过度表达在高剂量辐射后导致部分修复缺陷。恢复野生型，但不是D165 N Artemis的化疗/放射抗性表明，缺乏核酸内切修剪的DNA末端的Artemis缺陷细胞中的双链裂解剂的敏感性的主要原因。
Deficiency in Artemis is associated with lack of V(D)J recombination, sensitivity to radiation and radiomimetic drugs, and failure to repair a subset of DNA double-strand breaks (DSBs). Artemis harbors an endonuclease activity that trims both 5′- and 3′-ends of DSBs. To examine whether endonucleolytic trimming of terminally blocked DSBs by Artemis is a biologically relevant function, Artemis-deficient fibroblasts were stably complemented with either wild-type Artemis or an endonuclease-deficient D165N mutant. Wild-type Artemis completely restored resistance to γ-rays, bleomycin and neocarzinostatin, and also restored DSB-repair proficiency in G0/G1 phase as measured by pulsed-field gel electrophoresis and repair focus resolution. In contrast, cells expressing the D165N mutant, even at very high levels, remained as chemo/radiosensitive and repair deficient as the parental cells, as evidenced by persistent γ-H2AX, 53BP1 and Mre11 foci that slowly increased in size and ultimately became juxtaposed with promyelocytic leukemia protein nuclear bodies. In normal fibroblasts, overexpression of wild-type Artemis increased radioresistance, while D165N overexpression conferred partial repair deficiency following high-dose radiation. Restoration of chemo/radioresistance by wild-type, but not D165N Artemis suggests that the lack of endonucleolytic trimming of DNA ends is the principal cause of sensitivity to double-strand cleaving agents in Artemis-deficient cells.
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