Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5–Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5–Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5–Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5–Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5–Sae3 stabilized Dmc1 nucleating clusters with two to three molecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5–Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5–Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.

中文翻译：

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Mei5–Sae3 稳定 Dmc1 成核簇，可在 RPA 包被的单链 DNA 上高效组装 Dmc1


减数分裂中的同源间重组需要减数分裂特异性重组酶 Dmc1。在酿酒酵母中，Mei5-Sae3 复合物促进 Dmc1 加载到复制蛋白 A （RPA） 包被的单链 DNA （ssDNA） 上，以形成核蛋白丝。在体内，Dmc1 和 Mei5-Sae3 在染色体上的共定位是相互依赖的。然而，Mei5-Sae3 在介导 Dmc1 活性中的机制作用仍不清楚。我们使用单分子荧光共振能量转移和共定位单分子光谱实验来阐明 Mei5-Sae3 如何刺激 ssDNA 和 RPA 包被的 ssDNA 上的 Dmc1 组装。我们表明，Mei5-Sae3 通过优先降低 Dmc1 解离速率来稳定裸 DNA 上具有 2 到 3 个分子的 Dmc1 成核簇。Mei5–Sae3 还刺激 RPA 包被的 DNA 上的 Dmc1 组装。使用绿色荧光蛋白标记的 RPA，我们展示了在 RPA 解离之前 ssDNA 上 Dmc1 和 RPA 的中间体共存。此外，RPA 的置换效率取决于 Dmc1 浓度，其依赖性与 Dmc1 簇对短 ssDNA 的稳定性呈正相关。这些发现表明，Mei5-Sae3 通过稳定 Dmc1 成核簇介导 Dmc1 与 RPA 包被的 ssDNA 的结合，从而改变 DNA 上的 RPA 动力学以促进 RPA 解离。