School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China.
Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310003, China.
Adv Sci (Weinh). 2023 Oct;10(28):e2206931. doi: 10.1002/advs.202206931. Epub 2023 Aug 15.
In response to genotoxic stress-induced DNA damage, TopBP1 mediates ATR activation for signaling transduction and DNA damage repair. However, the detailed molecular mechanism remains elusive. Here, using unbiased protein affinity purification and RNA sequencing, it is found that TopBP1 is associated with pre-ribosomal RNA (pre-rRNA). Pre-rRNA co-localized with TopBP1 at DNA double-strand breaks (DSBs). Similar to pre-rRNA, ribosomal proteins also colocalize with TopBP1 at DSBs. The recruitment of TopBP1 to DSBs is suppressed when cells are transiently treated with RNA polymerase I inhibitor (Pol I-i) to suppress pre-rRNA biogenesis but not protein translation. Moreover, the BRCT4-5 of TopBP1 recognizes pre-rRNA and forms liquid-liquid phase separation (LLPS) with pre-rRNA, which may be the molecular basis of DSB-induced foci of TopBP1. Finally, Pol I-i treatment impairs TopBP1-associated cell cycle checkpoint activation and homologous recombination repair. Collectively, this study reveals that pre-rRNA plays a key role in the TopBP1-dependent DNA damage response.
在应对遗传毒性应激诱导的 DNA 损伤时，TopBP1 介导 ATR 的激活，从而进行信号转导和 DNA 损伤修复。然而，其详细的分子机制仍不清楚。在这里，我们采用无偏的蛋白亲和纯化和 RNA 测序技术，发现 TopBP1 与核糖体前体 RNA（pre-rRNA）相关联。pre-rRNA 与 TopBP1 在 DNA 双链断裂（DSBs）处共定位。与 pre-rRNA 相似，核糖体蛋白也与 TopBP1 在 DSB 处共定位。当细胞被短暂用 RNA 聚合酶 I 抑制剂（Pol I-i）处理以抑制 pre-rRNA 生物发生而不是蛋白翻译时，TopBP1 向 DSB 的募集受到抑制。此外，TopBP1 的 BRCT4-5 识别 pre-rRNA 并与 pre-rRNA 形成液-液相分离（LLPS），这可能是 DSB 诱导的 TopBP1 焦点形成的分子基础。最后，Pol I-i 处理会损害 TopBP1 相关的细胞周期检查点激活和同源重组修复。总之，这项研究揭示了 pre-rRNA 在依赖 TopBP1 的 DNA 损伤反应中起着关键作用。
