Super-resolution imaging of RAD51 and DMC1 in DNA repair foci reveals dynamic distribution patterns in meiotic prophase

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 (