ATM mediates repression of DNA end-degradation in an ATP-dependent manner
ATM mediates repression of DNA end-degradation in an ATP-dependent manner
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DOI：
10.1016/j.dnarep.2007.12.003
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发表时间：
2008-03-01
期刊：
影响因子：
3.8
通讯作者：
Dixon, Kathleen
中科院分区：
文献类型：
--
作者：
Rahal, Elias A.;Henricksen, Leigh A.;Dixon, Kathleen
共济失调毛细血管扩张突变(ATM)是一种与DNA双链断裂(DSB)修复和细胞周期调控相关的PI3-激酶样激酶(Pikk)。我们以前曾报道过ATM缺陷(A-T)和对照(ATM(+))细胞核提取液中DSB修复的类似效率；然而，与对照细胞相比，A-T核提取物的修复产物含有包含更长DNA片段的缺失。这些缺失似乎是由于微同源位点的末端连接造成的。这些数据表明，ATM阻碍了容易出错的修复途径，这些途径依赖于DNA末端的降解。这种降解可能解释了我们以前在A-T细胞提取液中观察到的更长的缺失。为了解决这种可能性，我们评估了DNA双链底物在A-T中的降解情况，并在DSB修复条件下控制了核提取液。我们观察到在A-T核提取物中信号强度从全长产物向较短产物的显著转变，并且在A-T核提取物中加入纯化的ATM可以恢复全长产物的检测。ATM对降解的这种抑制既依赖于ATP，也被Pikk抑制剂Wortmannin和咖啡因所抑制。在含有Pikk抑制剂的A-T核提取液中加入预磷酸化的ATM不足以抑制降解，这表明需要有激酶活性。这些结果表明，ATM在防止DNA末端降解方面的作用可能是通过抑制与微同源介导的末端连接有关的核酸酶来实现的。(C)2008爱思唯尔B.V.保留所有权利。
Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM(+)) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining. (c) 2008 Elsevier B.V. All rights reserved.