Chromosome Pairing Through Tensioned DNA Tethers Revealed by BRCA2 Meiotic Domain Deletion

Abstract

BRCA2 has multiple functional domains that interact with different partners, and is essential for both somatic and meiotic homologous recombination (HR). We created a Brca2{Delta}12-14 mouse 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). Brca2{Delta}12-14 mice 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 of Brca2{Delta}12-14, 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 in Brca2{Delta}12-14 meiocytes and controls, using super-resolution imaging of RPA and SYCP3 led to discovery of two novel features. First, in Brca2{Delta}12-14 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/561239v2_fig4.gif" ALT="Figure 4"> View larger version (41K): org.highwire.dtl.DTLVardef@1b95b8corg.highwire.dtl.DTLVardef@297466org.highwire.dtl.DTLVardef@198ef4borg.highwire.dtl.DTLVardef@1862be6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO Biochemical characterization of the DMC1-BRCA2 interaction. (A) BRCA2 regions deleted in the mice (yellow) and studied in pull-downs (black), HSF2BP-interacting motifs, PhePP motif and the peptide used in ref. 16 are indicated. Residue numbers for human BRCA2 are shown in black, mouse in blue. (B) GST pull-down with the indicated GST-BRCA2 fragments immobilized on GSH-sepharose beads and used to precipitate recombinant DMC1 or RAD51, followed by immunoblotting with anti-RAD51 antibody (cross-reacts with DMC1) and anti-GST antibodies. Full-size GST-fragment bands are indicated by red arrows, predicted Mw listed below the blot. Experiment was performed twice with same results. (C) Co-precipitation of purified recombinant untagged DMC1 and RAD51 with purified GST-tagged BRCA2 fragment F2s3 (wild type (wt) and F2406A variant (FA)) immobilized on the beads. Bound proteins were analyzed by SDS-PAGE and stained with Coomassie. (D) ITC analysis of the interaction between untagged DMC1 and synthetic peptides corresponding to BRCA2-F2s6 fragment, its F2406A variant, and the peptide from the RAD51-binding TR2 domain, containing a similar FxPP consensus. Experiments were performed twice with the same result, replicates are shown in Figure S5. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/561239v2_figS5.gif" ALT="Figure 5"> View larger version (21K): org.highwire.dtl.DTLVardef@1ac2377org.highwire.dtl.DTLVardef@4c710eorg.highwire.dtl.DTLVardef@924e49org.highwire.dtl.DTLVardef@151a83f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSupplementary Figure S5.C_FLOATNO (A) Repeat of the ITC experiment shown in Figure 4D. (B) GST pull-down using BRCA2-F2s3, the corresponding mouse BRCA2 fragment (I2327-Q2379), their variants with substitutions in the key phenylalanine (F2406A in human BRCA2 and F2351D in mouse BRCA2, to model the mutation introduced in the previously published mouse strain) and mouse or human his-DMC1. Proteins were expressed in E. coli, precipitated sequentially form crude lysates and detected by immunoblotting with anti-RAD51 and anti-GST antibodies. The experiment was done twice with the same result. C_FIG O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/561239v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1fcff59org.highwire.dtl.DTLVardef@e043a5org.highwire.dtl.DTLVardef@17924dcorg.highwire.dtl.DTLVardef@62fbc3_HPS_FORMAT_FIGEXP M_FIG C_FIG