Erasmus Optical Imaging Centre, Department of Pathology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands.
Department of Pathology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands.
PLoS Genet. 2020 Jun 5;16(6):e1008595. doi: 10.1371/journal.pgen.1008595. eCollection 2020 Jun.
The recombinase RAD51, and its meiosis-specific paralog DMC1 localize at DNA double-strand break (DSB) sites in meiotic prophase. While both proteins are required during meiotic prophase, their spatial organization during meiotic DSB repair is not fully understood. Using super-resolution microscopy on mouse spermatocyte nuclei, we aimed to define their relative position at DSB foci, and how these vary in time. We show that a large fraction of meiotic DSB repair foci (38%) consisted of a single RAD51 nanofocus and a single DMC1 nanofocus (D1R1 configuration) that were partially overlapping with each other (average center-center distance around 70 nm). The vast majority of the rest of the foci had a similar large RAD51 and DMC1 nanofocus, but in combination with additional smaller nanofoci (D2R1, D1R2, D2R2, or DxRy configuration) at an average distance of around 250 nm. As prophase progressed, less D1R1 and more D2R1 foci were observed, where the large RAD51 nanofocus in the D2R1 foci elongated and gradually oriented towards the distant small DMC1 nanofocus. D1R2 foci frequency was relatively constant, and the single DMC1 nanofocus did not elongate, but was frequently observed between the two RAD51 nanofoci in early stages. D2R2 foci were rare (<10%) and nearest neighbour analyses also did not reveal cofoci formation between D1R1 foci. However, overall, foci localized nonrandomly along the SC, and the frequency of the distance distributions peaked at 800 nm, indicating interference and/or a preferred distance between two ends of a DSB. DMC1 nanofoci where somewhat further away from the axial or lateral elements of the synaptonemal complex (SC, connecting the chromosomal axes of homologs) compared to RAD51 nanofoci. In the absence of the transverse filament of the SC, early configurations were more prominent, and RAD51 nanofocus elongation occurred only transiently. This in-depth analysis of single cell landscapes of RAD51 and DMC1 accumulation patterns at DSB repair sites at super-resolution revealed the variability of foci composition, and defined functional consensus configurations that change over time.
重组酶 RAD51 和其减数分裂特异性同源物 DMC1 在减数分裂前期定位于 DNA 双链断裂 (DSB) 位点。虽然这两种蛋白在减数分裂前期都需要，但它们在减数分裂 DSB 修复过程中的空间组织尚不完全清楚。本研究使用超分辨率显微镜研究了小鼠精母细胞核，旨在定义它们在 DSB 焦点中的相对位置，以及这些位置随时间的变化。我们发现，很大一部分减数分裂 DSB 修复焦点（38%）由一个 RAD51 纳米焦点和一个 DMC1 纳米焦点（D1R1 构型）组成，它们彼此部分重叠（平均中心-中心距离约为 70nm）。其余焦点的绝大多数具有类似的大 RAD51 和 DMC1 纳米焦点，但与其他较小的纳米焦点（D2R1、D1R2、D2R2 或 DxRy 构型）组合在一起，平均距离约为 250nm。随着前期的进展，观察到更少的 D1R1 焦点和更多的 D2R1 焦点，其中 D2R1 焦点中的大 RAD51 纳米焦点拉长并逐渐朝向远处的小 DMC1 纳米焦点。D1R2 焦点的频率相对稳定，单个 DMC1 纳米焦点没有拉长，但在早期阶段经常观察到两个 RAD51 纳米焦点之间。D2R2 焦点很少（<10%），最近邻分析也没有显示 D1R1 焦点之间的共焦点形成。然而，总的来说，焦点沿着 SC 非随机定位，距离分布的频率在 800nm 处达到峰值，表明两个 DSB 末端之间存在干扰和/或优选距离。与 RAD51 纳米焦点相比，DMC1 纳米焦点距离联会复合体（SC，连接同源染色体轴）的轴向或侧向元件稍远。在缺乏 SC 的横向丝的情况下，早期的构型更为突出，RAD51 纳米焦点的拉长仅短暂发生。这项对 RAD51 和 DMC1 在 DSB 修复位点的超分辨率单细胞景观的深入分析揭示了焦点组成的可变性，并定义了随时间变化的功能共识构型。
