Qin Caolitao, Wang Yun-Long, Zhou Jin-Ying, Shi Jie, Zhao Wan-Wen, Zhu Ya-Xi, Bai Shao-Mei, Feng Li-Li, Bie Shu-Ying, Zeng Bing, Zheng Jian, Zeng Guang-Dong, Feng Wei-Xing, Wan Xiang-Bo, Fan Xin-Juan
Henan Provincial Key Laboratory of Radiation Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China.
Department of Radiation Oncology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510655, P.R. China.
Nucleic Acids Res. 2023 Oct 13;51(18):9733-9747. doi: 10.1093/nar/gkad686.
RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 were not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, might be implicated. Recently, liquid-liquid phase separation (LLPS) has been characterized as a novel mechanism for the organization of key signaling molecules to drive their particular cellular functions. Here, we characterized that RAP80 LLPS at DSB was required for RAP80-mediated BRCA1 recruitment. Both cellular and in vitro experiments showed that RAP80 phase separated at DSB, which was ascribed to a highly disordered region (IDR) at its N-terminal. Meanwhile, the Lys63-linked poly-ubiquitin chains that quickly formed after DSBs occur, strongly enhanced RAP80 phase separation and were responsible for the induction of RAP80 condensation at the DSB site. Most importantly, abolishing the condensation of RAP80 significantly suppressed the formation of BRCA1 foci, encovering a pivotal role of RAP80 condensates in BRCA1 recruitment and radiosensitivity. Together, our study disclosed a new mechanism underlying RAP80-mediated BRCA1 recruitment, which provided new insight into the role of phase separation in DSB repair.
RAP80已被确定为BRCA1-A复合物的一个组成部分，负责将BRCA1招募到DNA双链断裂（DSB）处。然而，我们和其他人发现，RAP80和BRCA1的招募在时间上并非绝对同步，这表明除了物理相互作用外，可能还涉及其他机制。最近，液-液相分离（LLPS）已被确定为一种组织关键信号分子以驱动其特定细胞功能的新机制。在这里，我们发现DSB处的RAP80 LLPS是RAP80介导的BRCA1招募所必需的。细胞实验和体外实验均表明，RAP80在DSB处发生相分离，这归因于其N端的一个高度无序区域（IDR）。同时，DSB发生后迅速形成的K63连接的多聚泛素链强烈增强了RAP80的相分离，并导致RAP80在DSB位点凝聚。最重要的是，消除RAP80的凝聚显著抑制了BRCA1灶的形成，揭示了RAP80凝聚物在BRCA1招募和放射敏感性中的关键作用。总之，我们的研究揭示了RAP80介导的BRCA1招募的新机制，为相分离在DSB修复中的作用提供了新的见解。
