Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan.
Institute of Biochemical Sciences, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan.
Nucleic Acids Res. 2024 Oct 28;52(19):11768-11784. doi: 10.1093/nar/gkae780.
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.
减数分裂中的同源重组需要一种减数分裂特异性重组酶，即 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，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 Dmc1 在 ssDNA 和 RPA 包被的 ssDNA 上组装。我们表明，Mei5-Sae3 通过优先降低 Dmc1 解离速率来稳定裸 DNA 上具有两个到三个分子的 Dmc1 成核簇。Mei5-Sae3 还刺激 Dmc1 在 RPA 包被的 DNA 上组装。使用绿色荧光蛋白标记的 RPA，我们在 RPA 解离之前显示了 ssDNA 上存在与 Dmc1 和 RPA 共存的中间产物。此外，RPA 的置换效率取决于 Dmc1 浓度，并且其依赖性与 Dmc1 在短 ssDNA 上的成核簇的稳定性呈正相关。这些发现表明了一个分子模型，即 Mei5-Sae3 通过稳定 Dmc1 成核簇来介导 Dmc1 在 RPA 包被的 ssDNA 上的结合，从而改变 DNA 上的 RPA 动力学以促进 RPA 解离。
