BRCA2 regulates DMC1-mediated recombination through the BRC repeats

Abstract

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1-3 and 6-8. However, BRC1-3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6-8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1-ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPAssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.T he breast cancer susceptibility protein 2, BRCA2, regulates RAD51-mediated homologous recombination (HR) (1-3). Both RAD51, a DNA strand exchange protein, and its meiotic counterpart, DMC1 (disrupted meiotic cDNA 1 or DNA meiotic recombinase 1), promote HR through the formation of a nucleoprotein filament on ssDNA (4). This filament finds and invades a homologous template, resulting in a DNA strand invasion product called a joint molecule or a displacement-loop (D-loop). The joint molecule provides a primer template for the new DNA synthesis required to repair the DNA double strand break (DSB).The first evidence implicating BRCA2 in meiosis came from studies in Ustilago maydis, where strains lacking the BRCA2 ortholog, Brh2, resulted in absence of meiotic products (5). Shortly thereafter, mouse BRCA2 was inferred to coordinate the activities of RAD51 and DMC1 (6). The first direct interaction between BRCA2 and DMC1 was observed in plants (7) and later in humans (8). In the plant, Arabidopsis thaliana, the interaction between Brca2 and Dmc1 was mapped to the BRC repeats (9), a highly conserved motif comprising a sequence of ∼35 amino acids that is present at least once in all BRCA2-like proteins (10). In humans, BRCA2 contains eight BRC repeats that bind with different affinities to RAD51, and they segregate into two functional classes (11). Within a BRC repeat, two motifs that bind RAD51 have been identified: one comprising the consensus sequence FxxA that mimics the oligomerization interface (12) and contacts the catalytic domain of RAD51; the other binding module comprises the alpha-helical region o...

BRCA2 Hypomorphic Missense Variants Confer Moderate Risks of Breast Cancer

“…Full-length wildtype and mutant human BRCA2 proteins tagged with (N-terminal) green fluorescence protein (GFP) and maltose-binding protein (MBP) (GFP-MBP-BRCA2) were expressed and purified to near homogeneity (Supplementary Fig. S3) as described previously (18, 19). Full-length BRCA2 protein expression was confirmed by western blotting using an antibody against the C-terminus of BRCA2 (Supplementary Fig.…”

“…While purification of wildtype full-length BRCA2 protein to near homogeneity has previously been described (18, 19), in this study the functional integrity of purified mutant BRCA2 proteins was assessed for the first time in a quantitative ssDNA binding assay. Full-length proteins were used to limit potentially inaccurate interpretation of effects from partial protein fragments.…”

“…Wild-type and mutant human BRCA2 cDNAs were cloned into the C-terminal MBP-GFP-tagged phCMV1 expression plasmids and purified as described (18, 19). Briefly, 10 × 15-cm plates of HEK293 cells were transiently transfected using TurboFect (Thermo Scientific) following the manufacturer specifications and harvested 30 hours post-transfection.…”

“…Cell extracts were bound to Amylose resin (NEB), and the protein was eluted with 10 mM maltose. The eluate was further purified by ion exchange using BioRex 70 resin (BIO-RAD) and step eluted at 250mM, 450 mM, and 1M NaCl (18, 19). Each fraction was tested for nuclease contamination.…”

A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP

“…We next sought to understand how the HSF2BP pathogenic variant is mediating the observed meiotic alteration. HSF2BP has been shown to bind BRCA2, an essential protein for homologous meiotic recombination (Martinez et al, 2016;Sharan et al, 2004), in a direct manner tnatahrough its tryptophane at position 200 and the C-term (Gly2270-Thr2337) (Brandsma PhD thesis 2016;(Brandsma et al, 2019)). Given the impossibility to detect endogenous BRCA2 by IF in mouse spermatocytes, we carried out co-localization/interaction assays in a heterologous system by transfecting BRCA2-C (C-term) and HSF2BP in U2OS/HEK293T.…”