ABSTRACTThe recombinase RAD51, and its meiosis-specific paralog DMC1 localize at DNA double-strand break (DSB) repair sites in meiotic prophase nuclei. While both proteins are required during meiotic homologous recombination, 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%) contained a single RAD51 cluster and a single DMC1 cluster (D1R1 configuration) that were partially overlapping (average center-center distance around 70 nm). The majority of the rest of the foci had a similar combination of a major RAD51 and DMC1 cluster, but in combination with additional clusters (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 RAD51 cluster in the D2R1 foci elongated and gradually oriented towards the distant DMC1 cluster. This correlated with more frequently observed RAD51 bridges between the two DMC1 clusters. D1R2 foci frequency was more constant, and the single DMC1 cluster did not elongate, but was observed more frequently in between the two RAD51 clusters in early stages. D2R2 foci were rare (<10%) and nearest neighbour analyses also did not reveal pair formation between D1R1 foci. In the absence of the transverse filament of the synaptonemal complex (connecting the chromosomal axes of homologs), early configurations were more prominent, and RAD51 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 thus revealed the variability of foci composition, and defined functional consensus configurations that change over time.AUTHOR SUMMARYMeiosis is a specific type of cell division that is central to sperm and egg formation in sexual reproduction. It forms cells with a single copy of each chromosome, instead of the two copies that are normally present. In meiotic prophase, homologous chromosomes must connect to each other, to be correctly distributed between the daughter cells. This involves the formation and repair of double-strand breaks in the DNA. Here we used super-resolution microscopy to elucidate the localization patterns of two important DNA repair proteins: RAD51 and DMC1. We found that repair sites most often contain a single large cluster of both proteins, with or without one additional smaller cluster of either protein. RAD51 protein clusters displayed lengthening as meiotic prophase progressed. When chromosome pairing was disturbed, we observed changes in the dynamics of protein accumulation patterns, indicating that they actually correspond to certain repair intermediates changing in relative frequency of occurrence. These analyses of single meiotic DNA repair foci reveal the biological variability in protein accumulation patterns, and the localization of RAD51 and DMC1 relative to each other, thereby contributing to our understanding of the molecular basis of meiotic homologous recombination.
The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack