State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America.
PLoS Genet. 2019 Feb 4;15(2):e1007952. doi: 10.1371/journal.pgen.1007952. eCollection 2019 Feb.
Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.
减数分裂重组允许同源染色体之间交换遗传物质。复制蛋白 A (RPA) 复合物是主要的单链 DNA 结合复合物，几乎参与所有 DNA 代谢过程，但由于 RPA 突变小鼠的胚胎致死性，其在哺乳动物减数分裂重组中的作用仍不清楚。RPA 是由 RPA1、RPA2 和 RPA3 组成的异三聚体。我们发现，最大亚基 RPA1 的缺失导致 RPA2 和 RPA3 的消失，从而导致 RPA 复合物的缺失。使用可诱导的生殖系特异性失活策略，我们发现 RPA 的缺失完全阻断了 RAD51/DMC1 重组酶到程序性减数分裂 DNA 双链断裂的加载，从而阻断了染色体配对和联会所需的链入侵。令人惊讶的是，MEIOB、SPATA22 和 ATR 加载到 DNA 双链断裂是 RPA 非依赖性的，并且在没有 RPA 的情况下不会促进 RAD51/DMC1 的募集。最后，RPA 的失活减少了交叉形成。我们的结果表明，RPA 在减数分裂重组中发挥两个不同的作用：在早期阶段招募重组酶的必需作用，以及在后期促进交叉形成的重要作用。
