Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context
Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context
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DOI：
10.1074/jbc.m111.309252
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发表时间：
2011-12-09
影响因子：
4.8
通讯作者：
Stark, Jeremy M.
中科院分区：
文献类型：
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作者：
Gunn, Amanda;Bennardo, Nicole;Stark, Jeremy M.
在修复多个染色体双链断裂 (DSB) 过程中，匹配正确的 DSB 末端对于限制重排至关重要。为了研究正确最终用途的维持，我们检查了由核酸内切酶 I-SceI 和 3' 非进行性核酸外切酶 Trex2 生成的两个串联非粘性 DSB 的修复，这两个酶可以表达为 I-SceI-Trex2 融合体。我们检查了维持正确末端（近端-EJ）与使用不正确末端（远端-EJ）的末端连接（EJ）修复，这提供了不正确末端使用（远端使用）的相对测量。先前的研究表明，ATM 对于限制远端使用非常重要。在这里，我们表明 DNA-PKcs 激酶活性和 RAD50 对于限制远端使用也很重要，但 H2AX 是可有可无的。相比之下，我们发现 ATM、DNA-PKcs 和 RAD50 对需要末端处理的修复事件具有明显的影响。此外，我们开发了报告基因，使用诱导型启动子来检查转录环境对 DSB 修复的影响。我们发现，活性启动子下游的 DSB 显示出更高的远端使用频率，并且更依赖 ATM 来限制不正确的最终使用。相反，DSB 转录上下文不会影响 EJ 期间的末端加工、同源定向修复的频率或 RAD50 和 DNA-PKcs 在限制远端使用中的作用。我们认为，RAD50、DNA-PKcs 激酶活性和转录背景对于限制多个 DSB 的 EJ 修复期间的错误最终使用都很重要，但这些因素和条件在需要末端处理的修复事件期间具有不同的作用。
During repair of multiple chromosomal double strand breaks (DSBs), matching the correct DSB ends is essential to limit rearrangements. To investigate the maintenance of correct end use, we examined repair of two tandem noncohesive DSBs generated by endonuclease I-SceI and the 3' nonprocessive exonuclease Trex2, which can be expressed as an I-SceI-Trex2 fusion. We examined end joining (EJ) repair that maintains correct ends (proximal-EJ) versus using incorrect ends (distal-EJ), which provides a relative measure of incorrect end use (distal end use). Previous studies showed that ATM is important to limit distal end use. Here we show that DNA-PKcs kinase activity and RAD50 are also important to limit distal end use, but that H2AX is dispensable. In contrast, we find that ATM, DNA-PKcs, and RAD50 have distinct effects on repair events requiring end processing. Furthermore, we developed reporters to examine the effects of the transcription context on DSB repair, using an inducible promoter. We find that a DSB downstream from an active promoter shows a higher frequency of distal end use, and a greater reliance on ATM for limiting incorrect end use. Conversely, DSB transcription context does not affect end processing during EJ, the frequency of homology-directed repair, or the role of RAD50 and DNA-PKcs in limiting distal end use. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing.