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Comparative Study
. 2005 Jun 14;102(24):8537-42.
doi: 10.1073/pnas.0407266102. Epub 2005 Jun 3.

BRCA2 BRC motifs bind RAD51-DNA filaments

Vitold E Galkin  1 , Fumiko Esashi, Xiong Yu, Shixin Yang, Stephen C West, Edward H Egelman
Affiliations
Comparative Study

BRCA2 BRC motifs bind RAD51-DNA filaments

Vitold E Galkin et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Fig. 1.
Fig. 1.
Effect of BRC peptides on RAD51–DNA nucleoprotein filament formation. BRC3 (concentrations indicated) and RAD51 (1 μM) were incubated together before the addition of 32P-labeled linear dsDNA by using either ATP (a) or AMP-PNP (b and c). Various BRC peptides or mutant derivatives of BRC3 were incubated with RAD51, before addition of the labeled DNA, in the presence of AMP-PNP After 60-min incubation at 37°C, protein–DNA complexes were analyzed by agarose gel electrophoresis followed by autoradiography.
Fig. 2.
Fig. 2.
EM shows morphological differences in RAD51–DNA filaments after incubation with BRC3. Shown are electron micrographs of a control RAD51–AMP-PNP–DNA filament ([RAD51] = 7 μM) (a) and BRC3–RAD51–AMP-PNP–DNA filaments ([RAD51] = 7 μM, [BRC3] = 7 μM) (b). The strong striations in the RAD51–DNA filament (a) correspond to the helical pitch of this filament (≈75–130 Å). The binding of the BRC3 peptide to the RAD51 filament is suggested by the great reduction in the appearance of these striations (b). (Scale bar: 1,000 Å.)
Fig. 3.
Fig. 3.
Three-dimensional reconstructions of BRC3 (b) and B2-4 (c) peptides binding to RAD51–DNA filaments, with a pure RAD51–DNA filament reconstruction (a) shown as a control. The crystal structure of an intact ScRad51 subunit (17) is fit into the filaments. In the presence of BRC3 (b) or B2-4 (c), additional mass can be seen (arrows) associated with the N-terminal domain. A portion of the α-helix containing residues 146–156 in ScRad51 projects outside of the reconstructions (a, blue arrow), consistent with the suggestion that this region might be flexible in the filament (17). The concentrations used for these reconstructions were [RAD51] = 7 μM, [BRC3] = 7 μM(b), and [B2-4] = 3.5 μM (c).
Fig. 4.
Fig. 4.
Reconstructions of RAD51–DNA filaments ([RAD51] = 7 μM) in the presence of the BRC4 peptide ([BRC4] = 7 μM) (c and d), a control reconstruction of a pure RAD51–DNA filament (b), and a crystal structure of a yeast Rad51 filament (a) (17). The two reconstructions in the presence of BRC4 (c and d) were generated by sorting filament segments into different classes based on similarity. The main difference between these reconstructions involves RAD51's N-terminal domain, which is not seen in c, and shifted considerably in d from the position seen in the control filament (b). In b and d the RAD51 ATP-binding core is shown in magenta, and the N-terminal domain is in red. The rotated position of an N-terminal domain (19), used to fit a compressed RadA filament reconstruction (18), is shown in green (b). The BRC4 crystal fragment (9) is shown in blue (c and d).
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