MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, 510006, China.
Cell Death Differ. 2020 Nov;27(11):3162-3176. doi: 10.1038/s41418-020-0570-8. Epub 2020 Jun 3.
ATR is a master regulator of cell response to replication stress. Adequate activation of ATR is essential for preventing genome aberrance induced by replication defect. However, the mechanism underlying ATR activation is not fully understood. Here, we identify that RBMX is an ssDNA binding protein that orchestrates a novel pathway to activate ATR. Using super-resolution STORM, we observe that RBMX and RPA bind to adjacent but nonoverlapping sites on ssDNA in response to replication stress. RBMX then binds to and facilitates positioning of TopBP1, which activates nearby ATR associated with RPA. In addition, ATR activation by ssDNA-RBMX-TopBP1 is independent of ssDNA-dsDNA junction and 9-1-1 complex. ChIP-seq analysis reveals that RBMX/RPA are highly enriched on repetitive DNAs, which are considered as fragile sites with high replication stress. RBMX depletion leads to defective localization of TopBP1 to replication stressed sites and inadequate activation of ATR. Furthermore, cells with deficient RBMX demonstrate replication defect, leading to formation of micronuclei and a high rate of sister-chromatin exchange, indicative of genome instability. Together, the results identify a new ssDNA-RBMX-TopBP1 pathway that is specifically required for activation of ATR on repetitive DNAs. Therefore, RBMX is a key factor to ensure genome stability during replication.
ATR 是细胞对复制应激反应的主要调节剂。ATR 的充分激活对于防止复制缺陷引起的基因组异常是必不可少的。然而，ATR 激活的机制尚未完全了解。在这里，我们鉴定出 RBMX 是一种 ssDNA 结合蛋白，它协调了一种新的途径来激活 ATR。使用超分辨率 STORM，我们观察到 RBMX 和 RPA 在复制应激下结合到 ssDNA 上相邻但不重叠的位点。然后，RBMX 结合并促进 TopBP1 的定位，TopBP1 激活附近与 RPA 相关的 ATR。此外，ssDNA-RBMX-TopBP1 对 ATR 的激活不依赖于 ssDNA-dsDNA 连接和 9-1-1 复合物。ChIP-seq 分析显示，RBMX/RPA 在重复 DNA 上高度富集，重复 DNA 被认为是具有高复制应激的脆弱位点。RBMX 耗竭导致 TopBP1 向复制应激部位的定位缺陷和 ATR 激活不足。此外，RBMX 缺陷的细胞表现出复制缺陷，导致形成微核和姐妹染色单体交换率高，表明基因组不稳定。总之，这些结果确定了一种新的 ssDNA-RBMX-TopBP1 途径，该途径专门用于在重复 DNA 上激活 ATR。因此，RBMX 是确保复制过程中基因组稳定性的关键因素。
