Koornneef, L. | Search bioRxiv

bioRxiv2022

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

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.

developmental biology↗

bioRxiv2020

A cryptic BRCA2 repeated motif binds to HSF2BP oligomers with no impact on meiotic recombination

BRCA2 plays a prominent role in meiotic homologous recombination (HR). Loss of BRCA2 or several of its meiotic partners causes fertility defects. One of these partners, HSF2BP, was recently discovered as expressed physiologically in germline and ectopically produced in cancer cells. It has an N-terminal coiled coil motif involved in direct binding to the protein BRME1, and both HSF2BP and BRME1 are essential for meiotic HR during spermatogenesis. It also interacts through its C-terminal Armadillo (ARM) domain with a conserved region of BRCA2 of unknown function. We analyzed the structural properties and functional consequences of the BRCA2-HSF2BP interaction and tested the emerging model of its involvement in meiosis. We solved the crystal structure of the complex between the BRCA2 fragment that is disordered in solution and the HSF2BP dimeric ARM domain. This revealed two previously unrecognized BRCA2 repeats that each interact with one ARM monomer from two different dimers. BRCA2 binding triggers ARM tetramerization, resulting in a complex containing two BRCA2 fragments connecting two ARM dimers. The 3D structures of the BRCA2 repeats are superimposable, revealing conserved contacts between the BRCA2 residues defining the repeats and the HSF2BP residues lining the groove of the ARM. This large interface is responsible for the nanomolar affinity of the interaction, significantly stronger than any other measured interaction involving BRCA2. Deleting exon 12 from Brca2, encoding the first repeat, disrupted BRCA2 binding to HSF2BP in vitro and in cells. However, Brca2{Delta}12/{Delta}12 mice with the same deletion were fertile and did not show any meiotic defects, contrary to the prediction from the model positing that HSF2BP acts as a meiotic localizer of BRCA2. We conclude that the high-affinity interaction between BRCA2 and HSF2BP and the resulting HSF2BP oligomerization are not required for RAD51 and DMC1 recombinase localization to meiotic double strand breaks and for productive meiotic HR.

molecular biology↗