Department of Biological Sciences, Molecular and Computational Biology Section, University of Southern California, Los Angeles, California, United States of America.
Faculty of Health and Medicine, Lancaster Medical School, Lancaster University, Lancaster, United Kingdom.
PLoS One. 2022 Aug 2;17(8):e0271905. doi: 10.1371/journal.pone.0271905. eCollection 2022.
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.
共济失调毛细血管扩张突变和 RAD3 相关 (ATR) 激酶被 DNA 复制应激和各种形式的 DNA 损伤激活，包括 DNA 双链断裂 (DSBs)。ATR 的激活需要招募到损伤部位，因为两种已知的 ATR 激活剂之一，拓扑异构酶 II 结合蛋白 (TOPBP1) 或 Ewing 肿瘤相关抗原 1 (Ewing's tumor-associated antigen 1, ETAA1)，必须存在才能启动信号转导。在这里，我们利用我们最近建立的 Xenopus 卵提取物中的 DSB 介导的 ATR 激活 (DMAX) 系统来研究 TOPBP1 是如何被招募到 DSB 上的，以便激活 ATR。我们报告说，TOPBP1 仅在 DSB 上短暂存在，半衰期不到 10 分钟。我们还研究了 TOPBP1 与 MRE11-RAD50-NBS1 (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 的缺失会阻止 CtIP 招募到 DSB 上，并部分抑制 TOPBP1 的招募。CtIP 的缺失对 MRN 或 TOPBP1 的招募没有影响。ATM 激酶活性的缺失会阻止 CtIP 的招募，并增强 MRN 和 TOPBP1 的招募。这些发现表明，有依赖于 MRN 和独立于 MRN 的途径将 TOPBP1 招募到 DSB 上以激活 ATR。最后，我们发现 9-1-1 复合物和 MDC1 对于 TOPBP1 招募到 DSB 上都是可有可无的。
