Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1

Abstract

Germline mutations in BRCA2 predispose to hereditary breast cancers. BRCA2 protein regulates recombinational repair by interaction with RAD51 via a series of degenerate BRC repeat motifs encoded by exon 11 (BRCA2(996-2113)), and an unrelated C-terminal domain (BRCA2(3265-3330)). BRCA2 is also required for meiotic recombination. Here, we show that human BRCA2 binds the meiosis-specific recombinase DMC1 and define the primary DMC1 interaction site to a 26 amino-acid region (BRCA2(2386-2411)). This region is highly conserved in BRCA2 proteins from a variety of mammalian species, but is absent in BRCA2 from Arabidopsis thaliana, Caenorhabditis elegans, and other eukaryotes. We demonstrate the critical importance of Phe2406, Pro2408, and Pro2409 at the conserved motif (2404)KVFVPPFK(2411). This interaction domain, defined as the PhePP motif, promotes specific interactions between BRCA2 and DMC1, but not with RAD51. Thus, the RAD51 and DMC1 interaction domains on BRCA2 are distinct from each other, allowing coordinated interactions of the two recombinases with BRCA2 at meiosis. These results lead us to suggest that BRCA2 is a universal regulator of RAD51/DMC1 recombinase actions.

BRCA2: a universal recombinase regulator

“…The identification of this novel DMC1-interacting motif in BRCA2 was surprising as, based on the observed interactions between AtBRCA2 and AtDMC1, it was expected that interactions between hBRCA2 and hDMC1 would be mediated though one or more of the BRC motifs. Yeast two-hybrid analyses did reveal an association between DMC1 and the BRC4 motif of hBRCA2, but this was so weak that it could not be substantiated in pull-down experiments (Thorslund et al, 2007). Moreover, interactions could not be observed between hDMC1 and a larger fragment of BRCA2 containing all eight BRC motifs.…”

“…Furthermore, the BRC2 motif of AtBRCA2(IV) was found to interact with AtDMC1, whereas the binding to RAD51 was mediated through the BRC4 motif, indicating that the AtBRC motifs exhibit different specificities for the two recombinases (Dray et al, 2006). Details of the interactions that take place between human BRCA2 and DMC1 protein were recently described (Thorslund et al, 2007). Using a combination of yeast two-hybrid analysis, pull-downs with recombinant tagged fusion proteins, and peptide-arrays, the primary DMC1 interaction site on BRCA2 was mapped to a motif located at BRCA2 .…”

“…Using a semiquantitative yeast two-hybrid assay, it was estimated that a fragment containing BRC motifs 1-4 interacted approximately 5 Â stronger with RAD51 than a fragment containing BRC motifs 5-8 (Chen et al, 1998b), and that the affinity of BRC4 for RAD51 was 5 Â stronger than that seen with BRC1 . Others have reported yeast two-hybrid studies with individual BRC motifs showing strong interactions between BRC motifs 1-4 and RAD51, but saw only weak evidence for interactions between RAD51 and BRC motifs 5-8 (Thorslund et al, 2007).…”

“…Similarly, it was shown that a glutathione S-transferasetagged fragment of BRCA2 containing BRC1-2, and a fragment containing BRC3-5 interacted with RAD51, whereas a fragment containing BRC5-8 failed to pulldown RAD51 Thorslund et al, 2007). Hence, the ability of different BRC motifs to bind to RAD51 varies from one motif to another and detection of an interaction is dependent upon the method of analysis.…”

“…hDMC1 was also found to associate with the TR2 region of BRCA2, but this interaction was weak in comparison to that which takes place between TR2 and RAD51, and was not affected by phosphorylation of S3291 (Thorslund et al, 2007). The biological significance of this interaction may be minor as transgenic mice with C-terminal BRCA2 deletions fail to exhibit meiotic defects (McAllister et al, 2002;Atanassov et al, 2005).…”