Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Nucleic Acids Res. 2023 Nov 27;51(21):11717-11731. doi: 10.1093/nar/gkad856.
Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes. Here, using purified BCDX2 (RAD51BCD-XRCC2) and CX3 (RAD51C-XRCC3) complexes and in vitro reconstituted biochemical systems, we mechanistically dissect their functions in forming and protecting reversed forks. We show that both RAD51 paralog complexes lack fork reversal activities. Whereas CX3 exhibits modest fork protection activity, BCDX2 significantly synergizes with RAD51 to protect DNA against attack by the nucleases MRE11 and EXO1. DNA protection is contingent upon the ability of RAD51 to form a functional nucleoprotein filament on DNA. Collectively, our results provide evidence for a hitherto unknown function of RAD51 paralogs in synergizing with RAD51 nucleoprotein filament to prevent degradation of stressed replication forks.
叉头反转是一种保守的机制，用于防止停滞的复制叉崩溃。形成和保护反转叉是确保叉完整性和稳定性的两个关键步骤。RAD51 的五个同源物，即 RAD51B、RAD51C、RAD51D、XRCC2 和 XRCC3，与重组酶 RAD51 在序列和结构上具有相似性，它们在这些过程中起着定义不明确的机制作用。在这里，我们使用纯化的 BCDX2（RAD51BCD-XRCC2）和 CX3（RAD51C-XRCC3）复合物以及体外重建的生化系统，从机制上解析了它们在形成和保护反转叉中的功能。我们表明，这两种 RAD51 同源物复合物都缺乏叉头反转活性。虽然 CX3 表现出适度的叉保护活性，但 BCDX2 与 RAD51 显著协同作用，保护 DNA 免受 MRE11 和 EXO1 核酸酶的攻击。DNA 保护取决于 RAD51 在 DNA 上形成功能性核蛋白丝的能力。总的来说，我们的结果为 RAD51 同源物与 RAD51 核蛋白丝协同作用以防止应激复制叉降解的未知功能提供了证据。
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