MRN-dependent and independent pathways for recruitment of TOPBP1 to DNA double-strand breaks.
MRN-dependent and independent pathways for recruitment of TOPBP1 to DNA double-strand breaks.
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DOI：
10.1371/journal.pone.0271905
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发表时间：
2022
期刊：
影响因子：
3.7
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中科院分区：
文献类型：
--
作者：
共济失调毛细血管扩张症突变和RAD 3相关（ATR）激酶被DNA复制应激激活，也被各种形式的DNA损伤激活，包括DNA双链断裂（DSB）。损伤部位的募集不足以进行ATR活化，因为两种已知的ATR活化剂之一，拓扑异构酶II结合蛋白（TOPBP 1）或尤因肿瘤相关抗原1，也必须存在以启动信号传导。在这里，我们采用我们最近建立的DSB介导的ATR激活爪蟾卵提取物（DMAX）系统，以研究如何TOPBP 1招募到DSB，使它可以激活ATR。我们报告说，TOPBP 1只是暂时存在于DSBs，半衰期不到10分钟。我们还研究了TOPBP 1与MRE 11-RAD 50-NBS 1（MRN），CtBP相互作用蛋白（CtIP）和共济失调毛细血管扩张突变（ATM）蛋白网络之间的关系。MRN的缺失阻止了CtIP向DSB的募集，并部分抑制了TOPBP 1的募集。CtIP丢失对MRN或TOPBP 1募集无影响。ATM激酶活性的丧失阻止CtIP募集并增强MRN和TOPBP 1募集。这些发现表明，有MRN依赖性和独立的途径，招募TOPBP 1到DSB的ATR激活。最后，我们发现9-1-1复合物和MDC 1都是TOPBP 1招募到DSB的关键。
Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase is activated by DNA replication stress and also by various forms of DNA damage, including DNA double-strand breaks (DSBs). Recruitment to sites of damage is insufficient for ATR activation as one of two known ATR activators, either topoisomerase II-binding protein (TOPBP1) or Ewing’s tumor-associated antigen 1, must also be present for signaling to initiate. Here, we employ our recently established DSB-mediated ATR activation in Xenopus egg extract (DMAX) system to examine how TOPBP1 is recruited to DSBs, so that it may activate ATR. We report that TOPBP1 is only transiently present at DSBs, with a half-life of less than 10 minutes. We also examined the relationship between TOPBP1 and the MRE11-RAD50-NBS1 (MRN), CtBP interacting protein (CtIP), and Ataxia Telangiectasia mutated (ATM) network of proteins. Loss of MRN prevents CtIP recruitment to DSBs, and partially inhibits TOPBP1 recruitment. Loss of CtIP has no impact on either MRN or TOPBP1 recruitment. Loss of ATM kinase activity prevents CtIP recruitment and enhances MRN and TOPBP1 recruitment. These findings demonstrate that there are MRN-dependent and independent pathways that recruit TOPBP1 to DSBs for ATR activation. Lastly, we find that both the 9-1-1 complex and MDC1 are dispensable for TOPBP1 recruitment to DSBs.
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