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. 2007 May 15;104(20):8299-304.
doi: 10.1073/pnas.0702805104. Epub 2007 May 2.

Stabilization of RAD-51-DNA filaments via an interaction domain in Caenorhabditis elegans BRCA2

Mark I R Petalcorin  1 , Vitold E Galkin, Xiong Yu, Edward H Egelman, Simon J Boulton
Affiliations

Stabilization of RAD-51-DNA filaments via an interaction domain in Caenorhabditis elegans BRCA2

Mark I R Petalcorin et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in BRCA2 predispose individuals to breast cancer, a consequence of the role of BRCA2 in DNA repair. Human BRCA2 interacts with the recombinase RAD51 via eight BRC repeats. Controversy has existed, however, about whether the BRC interactions are primarily with RAD51 monomers or with the RAD51-DNA helical polymer, and whether there is a single interaction or multiple ones. We show here that the single BRC motif in the Caenorhabditis elegans BRCA2 homolog, CeBRC-2, contains two different RAD-51-binding regions. One of these regions binds only weakly to RAD-51-DNA filaments but strongly to RAD-51 alone and corresponds to the part of human BRC4 crystallized with RAD51. Injection of a peptide corresponding to this region into worms inhibits the normal formation of RAD-51 foci in response to ionizing radiation (IR). Conversely, peptides corresponding to the second region bind strongly to RAD-51-DNA filaments but do not bind to RAD-51 alone. Three-dimensional reconstructions from electron micrographs show that this peptide binds to the RAD-51 N-terminal domain, which has been shown to have a regulatory function. Injection of this peptide into worms before IR leads to a dramatic increase and persistence of IR-induced RAD-51 foci. This peptide also inhibits the RAD-51 ATPase activity, required for filament depolymerization. These results support a model where an interaction with RAD-51 alone is likely involved in filament nucleation, whereas a second independent interaction is involved in stabilization of RAD-51 filaments by BRCA2. The multiple interactions between BRCA2-like molecules and RAD51 provide insights into why mutations in BRCA2 lead to cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Electron microscopy of C. elegans RAD51–DNA filaments and their interaction with CeBRC-2. (a) In the presence of ATP, RAD51 forms filaments on single-stranded DNA that show the helical striations characteristic of all RecA-like filaments. (b) Incubation of RAD51–DNA filaments with full-length CeBRC-2 leads to massive aggregation of these filaments, with no free filaments found. The BRC 21–89 peptide induced a similar degree of aggregation (data not shown). (c–e) Three-dimensional reconstructions of pure RAD51–DNA filaments (c), and these filaments decorated with the BRC_60–89 peptide (d). A difference map (red density, e) between the BRC_60–89-RAD51–DNA filament (d) and the undecorated RAD51 filament (c) shows that BRC_60–89 is binding between the N-terminal domains of two adjacent RAD51 subunits. The difference map (e) is shown only for two subunits, as the slight difference in symmetry between the volumes in c and d requires that one look only at local differences. The atomic structure (28) of yeast RAD51 has been fit into the reconstructions in c–e, and each subunit is shown in a different color. (Scale bar: 1,000 Å.)
Fig. 2.
Fig. 2.
Retardation of RAD-51–DNA filament migration in gels by BRC_60–89. (a) An alignment of human BRC3 and BRC4 with the BRC region of CeBRC-2, made by using ClustalW (29). The arrow indicates where the BRC4 fragment complexed with the RAD51 core domain in a crystal was terminated (6), whereas 60–89 indicates the residues in the BRC_60–89 peptide. (b) Gel shifts were performed with 1 μM RAD-51 and 4 μM BRC peptide unless stated otherwise. Protein–DNA complexes were resolved in 0.5% agarose with or without addition of 5% glycerol. PC, polyprotein complexes that failed to enter the gel. To refine the region of CeBRC-2 that binds monomeric RAD-51, yeast two-hybrid analysis (c) and GST pull downs (d) with the indicated fusion proteins were performed as described (9). (c) Interaction in the yeast two-hybrid produces blue colonies in the LacZ reporter assay and growth on 3-aminotriazol (3AT). (d) One microgram of the indicated GST-fusions was incubated with 1 μg of RAD-51 for 30 min before GST-fusions were bound to glutathione beads and then washed extensively in binding buffer. Bound proteins were released from the beads in SDS loading buffer and then resolved on a 4–12% gradient gel and stained with Coomassie blue. The faster migrating band in the GST_BRC-1–60 lane is a proteolytic cleavage product.
Fig. 3.
Fig. 3.
Dominant negative effects of BRC_21–50 and BRC_60–89 on RAD-51 focus formation. BRC_21–50 blocks RAD51 focus formation, whereas BRC_60–89 results in persistent RAD-51 foci, after IR. N2(Wt) animals were microinjected with the indicated peptide (1 mg/ml), subjected to 75 Gy of γ-irradiation, and then assessed for RAD51 focus formation. (a) Representative images of fixed mitotic germ-line nuclei 2 h postinjection with 1 mg/ml peptide and a further 2 h untreated or subjected to γ-irradiation, and then immunostained with RAD-51 antibodies and counterstained with DAPI. Positive control was N2(Wt) worms microinjected with indicated peptide or injection buffer (–); negative controls were rad-51(lg08791) and brc-2(tm1086) worms. (b) Quantification of RAD-51 focus formation in a. The number of RAD-51 foci per nucleus was measured in 50 nuclei for each treatment/genotype. (c) Representative two-dimensional projections of RAD-51 foci on bivalent chromosomes in oocyte nuclei arrested at diakinesis of meiotic prophase I, 2 h postinjection with the indicated peptide and a further 5 h posttreatment with 75 Gy of γ-irradiation. (d) The percentage of −1, −2, and −3 (position in the germ line) oocyte nuclei positive for RAD-51 foci in animals of the indicated genotype subjected to the indicated treatment (as in c). Fifty nuclei were assessed for each treatment/genotype. (b and d) Error bars indicate SEM from three independent experiments.
Fig. 4.
Fig. 4.
BRC_60–89 reduces the rate of ATP hydrolysis by RAD-51. (a and b) Time course of ATP hydrolysis by RAD-51 alone or in the presence of BRC_21–50, BRC_60–89, or BRC_scram peptides. (c and d) The same ATPase assays were performed with the BRC_Wt (20–89) and BRC_Mut (14) (missing seven conserved BRC repeat residues) peptides. Reactions were performed in the presence of φX174 ssDNA (a and c) or φX174 dsDNA (b and d), with 4 mM Mg2+. Peptides were present at an 8-fold molar excess over RAD-51 ([RAD-51] = 1 μM). Error bars represent SEM from three independent experiments.
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