RPA and RAD51 ChIP-seq from B6 and B6xCAST F1 PRDM9-Humanized/CAST mouse testes

Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) and proceeds via binding of RPA, RAD51 and DMC1 to single-stranded DNA (ssDNA) substrates created after the formation of DSBs. Here, we report high-resolution in vivo maps of RPA and RAD51 binding in meiosis, mapping their binding locations and lifespans in a B6 and a genetically modified B6xCAST F1 mouse. We ascribe signals separately to the individual homologous chromosomes in the hybrid mouse, thereby separating the signal of binding to the chromosome where DSBs occurred and the chromosome that was used as template for repair. Together with super-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: whereas DMC1 binds near the break-site, RAD51 binds away from it. We characterize the D-loop, a critical intermediate bound by RPA, in vivo. These data show that DMC1, not RAD51, performs strand exchange in mammalian meiosis. We find that the localisation of D-loop intermediates is similar in crossover and non-crossover pathways, with a longer lifespan for crossover-destined intermediates. These findings answer long-standing questions about the molecular intermediates of recombination. Examination of RPA and RAD51 binding in the testes of C57BL/6J wild-type and (C57BL/6J x CAST/Eij)F1 PRDM9-Humanized/CAST mice

减数分裂重组由程序性DNA双链断裂（programmed DNA double-strand breaks, DSBs）启动，并通过RPA、RAD51与DMC1结合至DSB形成后产生的单链DNA（single-stranded DNA, ssDNA）底物而推进。本研究报道了减数分裂中RPA与RAD51结合的高分辨率体内图谱，解析了其在B6小鼠以及基因编辑B6×CAST F1小鼠中的结合位点与存留时长。我们将杂交小鼠中的信号分别分配至两条独立的同源染色体，由此区分了结合在发生DSB的染色体与作为修复模板的染色体上的信号。结合超分辨率显微镜技术与DMC1结合图谱，我们证实DMC1与RAD51在ssDNA上具有截然不同的空间定位：DMC1结合于断裂位点附近，而RAD51则结合于远离断裂位点的区域。我们在体内对RPA结合的关键重组中间体D环（D-loop）进行了表征。这些数据证实，在哺乳动物减数分裂过程中，执行链交换功能的是DMC1而非RAD51。我们发现，D环中间体在交换型（crossover）与非交换型（non-crossover）重组通路中的定位模式相似，但定向为交换型的中间体存留时长更长。这些发现解答了重组分子中间体领域长期存在的诸多科学问题。对C57BL/6J野生型小鼠以及(C57BL/6J × CAST/Eij)F1 PRDM9人源化/CAST小鼠睾丸中的RPA与RAD51结合情况进行检测