Hu Yingying, Chang Yen-Chan, Tsai You-Yang, Chang Hao-Yen, Chi Peter, Li Hung-Wen
Department of Chemistry, National Taiwan University, 10617 Taipei, Taiwan.
Institute of Biochemical Sciences, National Taiwan University, 10617 Taipei, Taiwan.
Nucleic Acids Res. 2025 Jul 19;53(14). doi: 10.1093/nar/gkaf676.
Homologous recombination is a key pathway for repairing DNA double-strand breaks with high fidelity. The assembly of recombinases on DNA to form nucleoprotein filaments is a crucial and tightly regulated step. This process requires the formation of a stable nucleus, followed by recombinase extension. The dynamic assembly and disassembly of recombinases directly affect recombination progression. Accessory proteins, such as SWI5-SFR1, regulate nucleation and extension steps, modulating filament stability and recombination efficiency. In this study, we extended our investigation to the rapid extension phase of RAD51 filament assembly. We found that mouse SWI5-SFR1 effectively reduces the dissociation probability of mouse RAD51 during filament extension, promoting more uniform filament growth. Step-size analysis revealed that mRAD51 assembles as various oligomeric units, with octamers being the predominant species. This observation reflects both the oligomeric nature and structural preference of mRAD51. In the presence of mSWI5-SFR1, the step-size distribution shifted toward tetramers, indicating that mSWI5-SFR1 modulates the oligomeric state of mRAD51 in solution, thereby facilitating extension and stabilizing DNA binding. Taken together, our findings bridge the gap between the nucleation and extension stages of filament assembly, and propose a comprehensive mechanism for RAD51 filament formation and its regulation by accessory proteins to ensure genome stability.
同源重组是一种以高保真度修复DNA双链断裂的关键途径。重组酶在DNA上组装形成核蛋白丝是一个关键且受到严格调控的步骤。这个过程需要形成一个稳定的核心，随后是重组酶的延伸。重组酶的动态组装和解离直接影响重组进程。辅助蛋白，如SWI5-SFR1，调节成核和延伸步骤，调节丝的稳定性和重组效率。在本研究中，我们将研究扩展到RAD51丝组装的快速延伸阶段。我们发现小鼠SWI5-SFR1有效地降低了小鼠RAD51在丝延伸过程中的解离概率，促进了更均匀的丝生长。步长分析表明，mRAD51以各种寡聚体形式组装，八聚体是主要形式。这一观察结果反映了mRAD51的寡聚性质和结构偏好。在mSWI5-SFR1存在的情况下，步长分布向四聚体转移，表明mSWI5-SFR1调节溶液中mRAD51的寡聚状态，从而促进延伸并稳定DNA结合。综上所述，我们的研究结果填补了丝组装成核和延伸阶段之间的空白，并提出了RAD51丝形成及其由辅助蛋白调控以确保基因组稳定性的全面机制。
