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. 2009 Mar 20;136(6):1032-43.
doi: 10.1016/j.cell.2009.02.019.

The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51

Aura Carreira  1 , Jovencio Hilario, Ichiro Amitani, Ronald J Baskin, Mahmud K K Shivji, Ashok R Venkitaraman, Stephen C Kowalczykowski
Affiliations

The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51

Aura Carreira et al. Cell. .

Abstract

The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, approximately 35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but not dsDNA, to stimulate DNA strand exchange. BRC4 acts by blocking ATP hydrolysis and thereby maintaining the active ATP-bound form of the RAD51-ssDNA filament. Single-molecule visualization shows that BRC4 does not disassemble RAD51-dsDNA filaments but rather blocks nucleation of RAD51 onto dsDNA. Furthermore, this behavior is manifested by a domain of BRCA2 comprising all eight BRC repeats. These results establish that the BRC repeats modulate RAD51-DNA interaction in two opposing but functionally reinforcing ways: targeting active RAD51 to ssDNA and prohibiting RAD51 nucleation onto dsDNA. Thus, BRCA2 recruits RAD51 to DNA breaks and, we propose, the BRC repeats regulate DNA-binding selectivity.

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Figures

Figure 1
Figure 1. Stimulation of RAD51-ssDNA complex formation by BRC4 and BRCA2BRC1-8
(A) Gel showing RAD51 (3 μM) incubated with either GST-BRC4 or GST-Δ1524-30 prior to incubation with 32P-labeled dT40 ssDNA (0.3 μM) for 1 h in the presence of ATP, Mg2+ and Ca2+ (B) Quantification of protein-DNA complexes with GST-BRC4 (filled circles) or GST-Δ1524-30 (open circles) obtained as in (A), using: ATP, Mg2+ + Ca2+ (●); ATP and Mg2+ (formula image); ADP, Mg2+ + Ca2+ (formula image), ATP and Ca2+ (formula image); AMP-PNP, Mg2+ + Ca2+ (formula image); or no nucleotide cofactor, Mg2+ + Ca2+ (formula image). Data obtained with the control mutant, GST-T1526A, ATP, Mg2+ + Ca2+: (formula image). (C) BRC4 forms a stoichiometric complex with RAD51-ssDNA complexes. RAD51 (0.15 μM) was incubated with BRC4 (formula image) or BRCA2BRC1-8 (formula image), or RAD51 (0.3 μM) was incubated with BRC4 (●), prior to incubation with 32P-labeled dT40 ssDNA (15 nM) for another 15 min in the presence of ATP, Mg2+ and Ca2+. Due to limitations of the protein stock, higher BRCA2BRC1-8 concentrations could not be examined. (D) BRC4 increases the stability of RAD51-ssDNA complexes to increasing concentrations salt: complexes were formed with RAD51 (0.9 μM) alone (upper panel) or RAD51 (0.9 μM) and GST-BRC4 (3.6 μM) (lower panel), in the presence of ADP and Mg2+.
Figure 2
Figure 2. BRC4 inhibits ATP hydrolysis and permits accumulation of ATP-bound RAD51-ssDNA filaments
(A) BRC4 reduces the rate of ATP hydrolysis by RAD51. RAD51 (3 μM) was incubated with increasing concentrations of GST-BRC4 (filled circles) or the control peptide GST-Δ1524-30 (open circles), as indicated, prior addition of dT40 (●), dT60 (formula image), dT90 (formula image), M13 ssDNA (formula image), or ϕX174 ssDNA (formula image), and further incubated for 1h in the presence of 0.5 mM ATP and 4 mM Mg2+. The dashed line represents the DNA-independent rate of ATP hydrolysis by RAD51 (~0.2 μM/min). (B) Nucleotides bound to purified nucleoprotein filaments as a function of BRC4 concentration as measured by TLC. (C) Amount of total nucleotide (ATP+ADP) bound to the RAD51-ssDNA complex formed with either dT40 (black circles) or ϕX174 ssDNA (orange circles) in presence of GST-BRC4 (filled circles) or GST-Δ1524-30 (open circles), relative to the amount in the absence of BRC4. (D) Percentages of ATP (solid lines) and ADP (dashed lines) in the RAD51 nucleoproteins filaments formed on dT40 (●) or ϕX174 ssDNA (formula image).
Figure 3
Figure 3. BRC4 and BRCA2BRC1-8 block RAD51-dsDNA complex formation
(A) Autoradiograph of an agarose gel showing RAD51 (3 μM) incubated with either GST-BRC4 or GST-Δ1524-30 prior to incubation with 32P-labeled ϕX174 linear dsDNA (5 μM, nt) and further incubation for 1 h in the presence of ATP, Mg2+ and Ca2+. (B) Quantification of data as in (A): in the presence of ATP, Mg2+ + Ca2+ (●); ADP, Mg2+ + Ca2+ (formula image); ATP and Ca2+ (formula image); or AMP-PNP, Mg2+ + Ca2+ (formula image). Filled circles and solid lines correspond to GST-BRC4; open circles and dashed lines correspond to GST-Δ1524-30. Data obtained with the control mutant, GST-T1526A, in the presence of ATP, Mg2+ + Ca2+ :(formula image). (C) The effect of BRC4 or BRCA2BRC1-8 on RAD51-dsDNA complex formation using dA40·dT40 dsDNA. RAD51 (0.15 μM) was incubated with GST-BRC4 or BRCA2BRC1-8, at the concentrations indicated prior to incubation with 32P-labeled dA40·dT40 dsDNA (2.4 μM, nt) 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. (D) Data obtained as in (C), quantified and plotted: GST-BRC4, (●); BRCA2BRC1-8 (formula image).
Figure 4
Figure 4. Single-molecule visualization reveals that BRC4 and BRCA2BRC1-8 inhibit nucleation of RAD51 on dsDNA
(A) Schematic representation of the experiment. The bead-DNA-YOYO-1 complex is trapped (Step 1) and then washed to dissociate YOYO-1 (Step 2). The λ DNA is incubated in the protein-channel with fluorescently-labeled RAD51, 1 mM ATP, 2 mM Ca(OAc)2, and the indicated amount of peptide (Step 3). The resulting RAD51 filament is moved back to the observation channel for analysis (Step 4). (B) Temporal series of images showing RAD51 nucleoprotein filament assembly on one DNA molecule. From left to right: Filament formation by RAD51 alone, with GST-BRC4 and with GST-Δ1524-30, respectively, at 4:1 molar ratio (peptide:RAD51) or (C) 2:1 and 0.5:1 ratios using BRC4 or BRCA2BRC1-8. (D) Cluster appearance as a function of time on λ DNA after incubation with 250 nM RAD51 and 1 μM (formula image), 0.5 μM (formula image), 250 nM (formula image), and 125 nM (formula image) GST-BRC4; or 0.5 μM (formula image) and 125 nM (formula image) BRCA2BRC1-8. The dashed lines represent the linear regressions of the data. (E) Rate of cluster formation vs. BRC4 or BRCA2BRC1-8 concentration; each time point is the average of 4–9 individual molecules analyzed as in D.
Figure 5
Figure 5. BRC4 does not promote the disassembly of RAD51-dsDNA filaments
(A) Temporal series of images showing disassembly of a RAD51 (250 nM) nucleoprotein filament with 2 mM Mg(OAc)2 and 1 mM ATP in all channels: left panel, RAD51; right panel, RAD51 with GST-BRC4 (1 μM). (B) Time-dependence of filament dissociation after incubation with RAD51 alone (black) or with GST-BRC4 (red). Each time point is the average of 3-4 individual molecules, and the lines represent fits of the data to a single exponential decay. The dotted line represents the starting length of the naked λ DNA.
Figure 6
Figure 6. BRC4 stimulates RAD51-mediated DNA strand exchange
(A) Diagram of DNA strand exchange between 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 GST-BRC4 or the control peptides GST-Δ1524-30 and GST-T1526A on DNA strand exchange between ϕX174 circular ssDNA and linear dsDNA promoted by RAD51 protein at 7.5 μM RAD51 (right panel); the uneven substrate bands resulted from a drying artifact. The left panel shows DNA strand exchange products at the optimal RAD51 concentration (3.75 μM). Size markers (kb) are shown in the left lane. (C) Quantification of the joint molecules (red) and nicked circular dsDNA (black) products using 3.75 μM RAD51 (inverted triangles); 7.5 μM RAD51 and GST-BRC4 (filled circles); 7.5 μM RAD51 and GST-Δ1524-30 (open circles); or 7.5 μM RAD51 and GST-T1526A (squares).
Figure 7
Figure 7. Model showing regulation of RAD51-DNA interactions by the BRC repeats
(A) The BRC repeats promote formation of the active ATP-RAD51-ssDNA filament at several steps: (1) In the presence of Mg2+-ATP, the BRC repeats promote binding of RAD51 to ssDNA. (2) By decreasing the rate of ATP hydrolysis, they permit accumulation of the active ATP-bound form of the filament. (3) The BRC repeats also stabilize the ADP-RAD51-ssDNA complex, permitting exchange of ADP for ATP and further increasing accumulation of an ATP-bound nucleoprotein filament. This filament is the active species in DNA strand exchange. (B) The BRC repeats prevent assembly of the non-productive ATP-RAD51-dsDNA filament: (4) The BRC repeats block nucleation of RAD51 onto dsDNA. (C) Proposed function of the intact BRCA2 protein in DSB repair (see text for details). A break in dsDNA is resected to produce ssDNA. RPA binds to the ssDNA, hindering RAD51 filament assembly. BRCA2 binds to the dsDNA-ssDNA junction, and loads RAD51 onto the ssDNA but blocks assembly onto the dsDNA. RAD51 filament growth extends the BRCA2-stablized nucleus, to form an ATP-bound nucleoprotein filament capable of homologous DNA pairing. Blue spheres represent a RAD51 monomer, T represents ATP, and D represents ADP; RPA is in orange, BRCA2 is brown, and the BRC repeats are represented in purple.
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