Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure

Abstract

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy to study how the two recombinases are organized on the ssDNA at individual DSBs, using a mouse model with severe defects in meiotic DSB formation and synapsis: Hormad1-/-. In accordance with previous analyses, we most frequently observed recombinase configurations with 1 DMC1 and 1 RAD51 nanofocus (D1R1), or two DMC1 nanofoci combined with a single RAD51 nanofocus (D2R1), and established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. In wild type, the D1R1:D2R1 frequency ratio decreased over time, but in the knockout, this ratio did not change due to increased D2R1 and decreased D1R1 foci frequency in leptotene. Also, the Hormad1-/- nanofoci were smaller. Nearest neighbor analysis of RAD51/DMC1 nanofoci showed two preferred distances at [~]300 and [~]900 nm in wild type, but only at [~]300 nm in Hormad1-/-. Combined with the lower total number of DSBs in the mutant, this suggests that a preferred distance of 900 nm represents the distance between DSB sites. We propose models with the D1R1 configuration representing a scenario with only one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or with both ends loaded by the two recombinases, but in below-resolution proximity. Surprisingly, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting the formation of coils in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. In conclusion, our data show that HORMAD1 affects the lifetime of recombinase-accumulation patterns at meiotic DSBs, as well as the structure of the synaptonemal complex. Author summaryMeiosis is a specialized cell division which generates haploid germ cells. In order to correctly pair homologous chromosomes in the first meiotic prophase, repair of programmed double strand breaks (DSBs) is essential. By unravelling molecular details of the protein assemblies at single DSBs, using super-resolution microscopy, we aim to understand the dynamics of repair intermediates and their functions. To this end, we investigated the localization of the two recombinases RAD51 and DMC1 in wild type and HORMAD1-deficient cells. HORMAD1 is involved in multiple aspects of homologous chromosome association: it regulates formation and repair of DSBs, and it stimulates formation of the synaptonemal complex, the macromolecular protein assembly that connects paired chromosomes. RAD51 and DMC1 enable chromosome pairing by promoting the invasions of the intact chromatids by single-stranded DNA ends that result from DSBs. We found that, in the absence of HORMAD1, RAD51 and DMC1 showed small but significant morphological and positional changes, combined with altered kinetics of specific RAD51/DMC1 configurations. We also observed changes in the structure of the synaptonemal complex in Hormad1-/ spermatocytes. This study contributes to a better understanding of the molecular details of meiotic homologous recombination and the role of HORMAD1 in meiotic prophase.