Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3′-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.

中文翻译：

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Rtt105 刺激 Rad51-ssDNA 组装并协调 Rad51 和 RPA 行动以促进同源重组修复


同源重组（HR）对于维持基因组稳定性至关重要。 HR 过程中，复制蛋白 A (RPA) 快速包被末端切除产生的 3' 尾单链 DNA (ssDNA)。然后，结合ssDNA的RPA必须及时被Rad51重组酶取代，形成Rad51核蛋白丝，驱动同源搜索和HR修复。细胞如何调节 Rad51 组装动态并协调 RPA 和 Rad51 行动以确保适当的 HR 仍然知之甚少。在这里，我们发现 Rtt105（Ty1 转座子调节剂）可刺激 Rad51 组装并在 HR 期间协调 RPA 和 Rad51 的活动。我们发现Rtt105在体外和体内与Rad51相互作用并抑制Rad51的腺苷5'三磷酸（ATP）水解活性。我们表明，Rtt105 在体外直接刺激动态 Rad51-ssDNA 组装、链交换和 D 环形成。值得注意的是，我们发现 Rtt105 通过不同的基序物理调节 Rad51 和 RPA 与 ssDNA 的结合，并且这两种调节对于促进 Rad51 成核、链交换和 HR 修复都是必要且上位的。因此，破坏任一相互作用都会损害 HR 并赋予 DNA 损伤敏感性，强调了 Rtt105 在协调 Rad51 和 RPA 作用中的重要性。我们的工作揭示了调节 Rad51 丝动力学和 HR 协调的额外机制。