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. 2011 Jun 28;108(26):10448-53.
doi: 10.1073/pnas.1106971108. Epub 2011 Jun 13.

Two classes of BRC repeats in BRCA2 promote RAD51 nucleoprotein filament function by distinct mechanisms

Aura Carreira  1 , Stephen C Kowalczykowski
Affiliations

Two classes of BRC repeats in BRCA2 promote RAD51 nucleoprotein filament function by distinct mechanisms

Aura Carreira et al. Proc Natl Acad Sci U S A. .

Abstract

The human tumor suppressor protein BRCA2 plays a key role in recombinational DNA repair. BRCA2 recruits RAD51 to sites of DNA damage through interaction with eight conserved motifs of approximately 35 amino acids, the BRC repeats; however, the specific function of each repeat remains unclear. Here, we investigated the function of the individual BRC repeats by systematically analyzing their effects on RAD51 activities. Our results reveal the existence of two categories of BRC repeats that display unique functional characteristics. One group, comprising BRC1, -2, -3, and -4, binds to free RAD51 with high affinity. The second group, comprising BRC5, -6, -7, and -8, binds to free RAD51 with low affinity but binds to the RAD51-ssDNA filament with high affinity. Each member of the first group reduces the ATPase activity of RAD51, whereas none of the BRC repeats of the second group affects this activity. Thus, through different mechanisms, both types of BRC repeats bind to and stabilize the RAD51 nucleoprotein filament on ssDNA. In addition, members of the first group limit binding of RAD51 to duplex DNA, where members of the second group do not. Only the first group enhances DNA strand exchange by RAD51. Our results suggest that the two groups of BRC repeats have differentially evolved to ensure efficient formation of a nascent RAD51 filament on ssDNA by promoting its nucleation and growth, respectively. We propose that the BRC repeats cooperate in a partially redundant but reinforcing manner to ensure a high probability of RAD51 filament formation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The individual BRC repeats of BRCA2 form complexes with RAD51, with distinct classes of affinities. (A) Each GST-tagged BRC peptide was tested individually for binding to RAD51 in a GST pull-down assay: RAD51 was incubated with the GST-BRC repeats, captured using glutathione beads, washed extensively to remove unbound proteins, analyzed by SDS/PAGE, and stained with SYPRO ORANGE. Lanes 1–4 contain increasing amounts of RAD51 (0.1, 0.2, 04, 0.8 μg) to generate a standard curve. Lane 5 or 6 is a control that contains the highest concentration of RAD51 used in the pull-down (2.4 μg), but in the absence of BRC peptide (for gels with GST-BRC1, -3, -4, and -6, the “RAD51 only” control is lane 6; lane 5 is empty in these gels). The next lane (6 or 7) is a control showing the BRC peptide, “BRCx only”, (0.5 μg) used in the pull-down experiment in the absence of RAD51. Lanes 7–10 (or 8–11) show increasing amounts of RAD51 incubated with each BRC peptide. Lanes 11–14 (or 12–15) contain increasing amounts of each BRC peptide (0.1, 0.2, 0.4, 0.5 μg) to generate a standard curve for the BRC peptide. The gel with GST-BRC8 contains an extra empty lane between lanes 10 and 11. An asterisk denotes the band that corresponds to contaminating GST (the amount of free GST could vary between different preparations of the same peptide). (B) The data from A were fitted to a single-site binding curve (Prism 5.0b), and are shown as solid lines. Error bars represent the standard deviation (SD) for two or more independent experiments. The data for BRC repeats 6–8 could not be fit accurately, and are shown connected by dashed lines.
Fig. 2.
Fig. 2.
Only BRC1, -2, -3 and -4 inhibit ssDNA-dependent ATP hydrolysis by RAD51. (A) BRC1, -2, -3, and -4: RAD51 (3 μM) was incubated with increasing concentrations of GST-BRC peptide, as indicated, prior to addition of dT40 ssDNA and was further incubated for 1 h in the presence of 0.5 mM ATP and 4 mM Mg2+. B. Same as A but using BRC5, -6, -7, or -8. Error bars represent the SD for two or more independent experiments; where not seen, the error bars are smaller than the data points.
Fig. 3.
Fig. 3.
Each of the BRC repeats stimulates RAD51-ssDNA complex formation. (A) Autoradiograph of an acrylamide gel showing RAD51 (3 μM) incubated with either GST-BRC1 (lanes 3–6) or GST-BRC2 (lanes 7–10) prior to incubation with 32P-labeled dT40 ssDNA (0.3 μM) for 1 h in the presence of ATP, Mg2+, and Ca2+. Lane 1 contains DNA alone. Lane 2 contains RAD51 and DNA, in the absence of BRC peptide. (B) Same as in A but with GST-BRC3 (lanes 3–6) or GST-BRC4 (lanes 7–10). (C) Same as in A but with GST-BRC5 (lanes 3–6) or GST-BRC6 (lanes 7–10). (D) Same as in A but with GST-BRC7 (lanes 3–6) or GST-BRC8 (lanes 7–10). (E) Quantification of RAD51 binding to ssDNA in the presence of increasing concentrations of BRC1, -2, -3, and -4 calculated from the data shown in A and B. F. Quantification of RAD51 binding to ssDNA in the presence of increasing concentrations of BRC5, -6, -7, and -8 calculated from the data shown in C and D. Error bars represent the SD for two or more independent experiments; where are not seen, the error bars are smaller than the data points.
Fig. 4.
Fig. 4.
BRC1, -2, -3, and -4 prevent formation of RAD51-dsDNA complexes. (A) EMSA analysis showing RAD51 (3 μM) incubated with GST-BRC1 prior to incubation with 32P-labeled dA40·dT40 dsDNA (0.3 μM, bp) and further incubation for 1 h in the presence of ATP, Mg2+, and Ca2+. Protein-DNA complexes were resolved in 6% PAGE and analyzed by autoradiography. Lane 1 contains DNA alone; lane 2 contains RAD51 incubated with DNA in the absence of BRC peptide. Lanes 3–8 contain the indicated concentration of BRC1 peptide and 3 μM RAD51. Lane 9 contains the maximum concentration of BRC1 peptide and DNA in the absence of RAD51. Lane 10 contains the indicated concentration of BRC4 and RAD51 incubated with DNA under the same conditions used with BRC1. (B) Same as in A but using BRC2 instead of BRC1. (C) Same as in A but using BRC3 instead of BRC1. (D) Quantification of data in A, B, and C: BRC1 (green), BRC2 (orange), BRC3 (blue), and BRC4 (purple; gel shown in Fig. S3). Error bars indicate SD from at least three independent experiments.
Fig. 5.
Fig. 5.
Only BRC-1, -2, -3 and -4 can stimulate the DNA strand exchange activity of RAD51. (A) Scheme of DNA strand exchange between ϕX174 circular ssDNA and homologous linear dsDNA to produce joint molecules (JM) and nicked circular dsDNA (NC). The asterisk shows the 32P-label on each strand. (B) Effect of BRC1 (lanes 2–6), BRC2 (lanes 10–14), BRC3 (lanes 15–19), or BRC4 (lanes 22–26) on DNA strand exchange with excess RAD51 (7.5 μM); lane 8 shows DNA strand exchange at the optimal RAD51 concentration (3.75 μM). (C–F) Quantification of the joint molecules (JM, filled symbols) and nicked circular dsDNA (NC, open symbols) products using 3.75 μM RAD51 (red squares, same in every graph) or 7.5 μM RAD51 (circles) and GST-BRC1 (C), BRC2 (D), BRC3 (E), or BRC4 (F). Error bars indicate SD from at least three independent experiments.
Fig. 6.
Fig. 6.
Proposed model for the role of the BRC repeats in the context of the entire BRCA2 protein in DSB repair (see text for details). Upon formation of a DSB, the dsDNA is resected to generate ssDNA tails. RAD51 binds to BRCA2 through the high-affinity BRC repeats, BRC1-4 (1); via this interaction, the BRC repeats alter the conformation of RAD51, enhancing ssDNA binding and slowing dsDNA binding. The binding of BRCA2 to ssDNA directs RAD51 onto the ssDNA of a processed DSB and restricts assembly onto the dsDNA nearby; the new conformation imposed by BRC1-4 on RAD51 (“locked” RAD51) allows nucleation onto ssDNA and stabilizes the nascent nucleoprotein filament by limiting the ATP hydrolysis by RAD51 (2). After nucleation, BRC repeats 5–8 bind the nascent RAD51 nucleoprotein filament and further stabilize filament extension locally (3). The subsequent BRCA2-independent growth of the RAD51 nucleoprotein filament displaces RPA from the ssDNA. At this point, BRCA2 can be released from the DNA to promote RAD51 nucleation at another DSB (4). The RAD51 filament continues to grow from the BRCA2-stablized nucleus to form the ATP-bound nucleoprotein filament capable of homologous DNA pairing (5).
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