Chromosome Pairing Through Tensed DNA Tethers Model Revealed by BRCA2 Meiotic Domain Deletion
Authors: Koornneef Lieke , van Rossum-Fikkert Sari E. , Sleddens-Linkels Esther , Miron Simona , Maas Alex , van Loon Yvette , Barchi Marco , Zinn-Justin Sophie , Essers Jeroen , Kanaar Roland , Baarends Willy M. , Zelensky Alex N. 
Published: October 2023

ABSTRACTBRCA2 has multiple functional domains that interact with different partners, and is essential for both somatic and meiotic homologous recombination (HR). We created aBrca2Δ12-14mouse model with an internal deletion of the region which we named “the meiotic domain of BRCA2”, as its loss results in complete failure of meiotic HR, while somatic HR is intact. The deletion in the protein includes the HSF2BP-binding motifs (exons 12-13) and the DMC1-binding PhePP domain (exon 14).Brca2Δ12-14mice showed complete infertility in both males and females, with sexually dimorphic features. Recombinase foci (both RAD51 and DMC1) were completely undetectable in mutant spermatocytes, but while DMC1 foci were also absent in mutant oocytes, RAD51 foci numbers were only partially reduced. The function of the PhePP domain for meiotic HR is unclear, but both the phenotype ofBrca2Δ12-14, and our biochemical data indicate that, along with the BRC repeats of BRCA2, PhePP is both critical and specific for DMC1 loading in meiotic HR, analogous to the C-terminal RAD51-specific TR2/CTRB. Further investigation of DSB end processing inBrca2Δ12-14meiocytes and controls, using super-resolution imaging of RPA and SYCP3 led to discovery of two novel features. First, in Brca2Δ12-14 oocytes, but not in the spermatocytes nor wild types, we observed RPA foci as doublets ∼200 nm apart, which could represent DSB end resolution into separate nanofoci. Second, we describe RPA structures that are completely HR-dependent and are indicative of long, double-stranded DNA connections between homologs prior to synapsis. Our observations lend support to a model for chromosome alignment via multiple HR-dependent DNA tethers that connect homologs and may be tensed. We propose that tether shortening (e.g. by dynamic adjustment of chromatin loops by meiotic cohesins) provides a plausible molecular mechanism to juxtapose homologs and initiate synapsis.Version 2 Revision SummaryThe main difference compared to version 1 (deposited on October 6 2023) is a more concise and structured description of the tensed DNA tether model of meiotic chromosome pairing, based on the discussions with colleagues and one round of peer review. In the new model presentation, we explicitly separated the inferences from the presented data from the two hypothetical propositions: (1) tether shortening contributes to pairing rather than simply accompanies it, and (2) the apparent tension, which reveals the tethers on chromosome spreads, also exists in the nuclei. We also clarified the definition of the tether, avoiding the ambiguous “RPA tether” term, and provided a more complete overview of the relevant prior literature on proteinaceous bridges and DNA connections. Biochemical data (Fig. 4, S5) has been replicated under uniform conditions and extended to mouse proteins. Manuscript has been reformatted to improve on-screen readability.