BRCA2 BRC motifs bind RAD51–DNA filaments

Abstract

Germ-line mutations in BRCA2 account for approximately half the cases of autosomal dominant familial breast cancers. BRCA2 has been shown to interact directly with RAD51, an essential component of the cellular machinery for homologous recombination and the maintenance of genome stability. Interactions between BRCA2 and RAD51 take place by means of the conserved BRC repeat regions of BRCA2. Previously, it was shown that peptides corresponding to BRC3 or BRC4 bind RAD51 monomers and block RAD51-DNA filament formation. In this work, we further analyze these interactions and find that at lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4. These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. The results provide insight into why mutation in just one of the eight BRC repeats would affect the way that BRCA2 protein interacts with the RAD51 filament. Disruption of a single RAD51 interaction site, one of several simultaneous interactions occurring throughout the BRC repeat-containing exon 11 of BRCA2, might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer.electron microscopy ͉ nucleoprotein filaments ͉ recombination ͉ genome stability and dynamics ͉ structural biology M utations in BRCA2 are associated with an increased risk of breast and ovarian cancers, and almost half the cases of inherited early onset breast cancers have been linked to mutations in BRCA2. The product of this gene, BRCA2 protein (384 kDa), interacts with RAD51, and it has been shown that both proteins are essential for homologous recombination, DNA repair, and the maintenance of genome stability. There appear to be a number of different interactions between BRCA2 and RAD51. One such interaction involves a C-terminal region of BRCA2 (1). In addition, BRCA2 interacts with RAD51 through the eight conserved BRC repeats in BRCA2 (2, 3), and mutations within these repeats are associated with cancer predisposition. The deletion of several BRC repeats in mice leads to cancer (4), and somatic mutations in BRC repeats have been found to be associated with breast cancer (5). Although there are eight BRC repeats in human BRCA2, in other organisms the number of BRC repeats is quite variable, with 15 found in a Trypanosoma BRCA2-like protein (6) and only one found in the Ustilago Brh2 protein (7).Many details of the interactions between BRCA2 and RAD51 are unclear, such as whether BRCA2 binds to the N-terminal domain of RAD51 (8) or to the nucleotide-binding core (3, 9). Recently, a fusion protein containing the nucleotide-binding core of RAD51 and a single BRC repeat was crystallized. Analysis of the x-ray structure of the fusion led t...

Interaction between Arabidopsis Brca2 and Its Partners Rad51, Dmc1, and Dss1

“…In mammals, the BRC repeats are involved in the interaction of Brca2 with Rad51 (Wong et al, 1997;Chen et al, 1998b;Davies et al, 2001;Galkin et al, 2005). Since an interaction between Brca2 and Dmc1 has never been described in any other organism than Arabidopsis (to our knowledge), there is no clue about the region of Brca2 that is involved in binding to Dmc1.…”

“…Yang et al (2002) demonstrated that a DNA-binding domain is present in the 800-amino acid C-terminal region of Brca2 that can stimulate Replication Protein A (RPA)-dependent strand transfer between homologous DNA molecules by Rad51; this region could only be crystallized in the presence of Dss1, which allowed the authors to define domains in this C-terminal region of Brca2 that were involved in binding Dss1. Work from Galkin et al (2005) also indicates that the BRC motifs number 3 and number 4 in human Brca2 can form stable complexes with Rad51-DNA nucleoprotein filaments. They also reveal the regions in Rad51 that are involved in contacting the BRC motifs.…”

BRCA2: a universal recombinase regulator

“…The BRC4 peptide was found to disrupt RAD51 filaments, presumably by binding RAD51 monomers in solution, thereby shifting the equilibrium towards BRC4-RAD51 heterodimer formation (Davies and Pellegrini, 2007;Esashi et al, 2007). However, this disruptive effect was only observed when BRC4 was present in molar excess compared to RAD51, and at lower concentrations of BRC4 it was found to associate with RAD51 filaments formed on dsDNA in the presence of the non-hydrolysable ATP analogue 5 0 -adenylyl-b, g-imidodiphosphate (AMP-PNP) (Galkin et al, 2005). When similar experiments were carried out with BRC3, it was found that excess BRC3 could again dissociate RAD51 filaments, but only in the presence of ATP, while sub-stoichiometric amounts of BRC3 were found to form stable associations with the RAD51 filaments.…”

“…When similar experiments were carried out with BRC3, it was found that excess BRC3 could again dissociate RAD51 filaments, but only in the presence of ATP, while sub-stoichiometric amounts of BRC3 were found to form stable associations with the RAD51 filaments. In the presence of a non-hydrolysable ATP analogue, BRC3 could form stable associations with the RAD51 filaments even when in excess over RAD51 (Galkin et al, 2005). That the different BRC motifs are non-equivalent in their ability to interact with RAD51 is further strengthened by the observation that a single mutation in RAD51-A190V prevents RAD51 interaction with BRC3 but not BRC4 .…”

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

“…The eight BRC repeats share only a core consensus sequence and appear to bind Rad51 with different affinities (10,22,25). In addition, BRC3 and BRC4, which have only 30% identity, appear to interact with Rad51 nonequivalently, with BRC3 binding to the N-terminal domain and BRC4 binding to the nucleotide-binding core domain (25,26).…”