Department of Chemistry, National Taiwan University, 10617 Taipei, Taiwan.
Institute of Biochemical Sciences, National Taiwan University, 10617 Taipei, Taiwan.
Proc Natl Acad Sci U S A. 2020 May 26;117(21):11257-11264. doi: 10.1073/pnas.1920368117. Epub 2020 May 13.
Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.
Dmc1 重组酶对于减数分裂中的同源重组至关重要。在这里，我们使用单分子系绳粒子运动实验和体外生化测定研究了 Dmc1 的核蛋白丝组装的动力学。由于成核步骤的动力学大大减少，ScDmc1 核蛋白丝不如 ScRad51 稳定。与 ScRad51 相比，ScDmc1 的单链 DNA（ssDNA）亲和力较低，导致其成核速率较低。令人惊讶的是，ScDmc1 主要在含有单链和双链 DNA 接头的 DNA 结构上引发，允许在 5'-3'极性上进行延伸，而 ScRad51 的引发强烈依赖于 ssDNA 长度。这种引发偏好对于哺乳动物 RAD51 和 DMC1 也是保守的。此外，ScDmc1 的引发可以被短的 ScRad51 补丁刺激，但不能被 EcRecA 刺激。下拉实验也证实了 ScDmc1 在溶液中与 ScRad51 的物理相互作用，但与 EcRecA 没有相互作用。我们的结果与一个模型一致，即 Dmc1 的引发可以通过结构成分（如 DNA 接头和蛋白质-蛋白质相互作用）和 DNA 极性来促进。它们提供了直接证据，证明了 Rad51 对于减数分裂重组的必要性，并突出了 Dmc1 核蛋白丝组装中的调控策略。
