ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the presence of ATP, 2) complementary DNA strand-exchange, 3) ATP hydrolysis transforming the RAD51 filament into an ADP-bound disassembly-competent state, and 4) RAD51 disassembly to provide access for DNA repairing enzymes. Of these steps, filament dynamics between the ATP- and ADP-bound states, and the RAD51 disassembly mechanism, are poorly understood due to the lack of near-atomic-resolution information of the ADP-bound RAD51–DNA filament structure. We report the cryo-EM structure of ADP-bound RAD51–DNA filaments at 3.1 Å resolution, revealing a unique RAD51 double-filament that wraps around ssDNA. Structural analysis, supported by ATP-chase and time-resolved cryo-EM experiments, reveals a collapsing mechanism involving two four-protomer movements along ssDNA for mechanical transition between RAD51 single- and double-filament without RAD51 dissociation. This mechanism enables elastic change of RAD51 filament length during structural transitions between ATP- and ADP-states.

ATP 依赖性 RAD51 重组酶通过催化四步过程在真核同源重组中发挥重要作用：1) 在 ATP 存在下在 ssDNA 上形成 RAD51 单丝组装，2) 互补 DNA 链交换，3) ATP水解将 RAD51 丝转化为 ADP 结合的解体状态，4) RAD51 解体为 DNA 修复酶提供通路。在这些步骤中，由于缺乏 ADP 结合的 RAD51-DNA 细丝结构的近原子分辨率信息，人们对 ATP 和 ADP 结合状态之间的细丝动力学以及 RAD51 分解机制知之甚少。我们以 3.1 Å 分辨率报道了 ADP 结合的 RAD51-DNA 丝的冷冻电镜结构，揭示了包裹 ssDNA 的独特 RAD51 双丝。在 ATP 追踪和时间分辨冷冻电镜实验的支持下，结构分析揭示了一种折叠机制，涉及沿 ssDNA 的两个四原聚体运动，用于 RAD51 单丝和双丝之间的机械转变，而无需 RAD51 解离。这种机制使得 RAD51 丝长度在 ATP 和 ADP 状态之间的结构转变期间能够弹性变化。