The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange

Abstract

The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2([BRC1-8])). Here, we show that BRCA2([BRC1-8]) exerts opposing effects on the binding of RAD51 to single-stranded (ss) versus double-stranded (ds) DNA substrates, enhancing strand exchange. BRCA2([BRC1-8]) alters the electrophoretic mobility of RAD51 bound to an ssDNA substrate, accompanied by an increase in ssDNA-bound protein assemblies, revealed by electron microscopy. Single-molecule fluorescence spectroscopy shows that BRCA2([BRC1-8]) promotes RAD51 loading onto ssDNA. In contrast, BRCA2([BRC1-8]) has a different effect on RAD51 assembly on dsDNA; it suppresses and slows this process. When homologous ssDNA and dsDNA are both present, BRCA2([BRC1-8]) stimulates strand exchange, with delayed RAD51 loading onto dsDNA accompanying the appearance of joint molecules representing recombination products. Collectively, our findings suggest that BRCA2([BRC1-8]) targets RAD51 to ssDNA while inhibiting dsDNA binding and that these contrasting activities together bolster one another to stimulate HDR. Our work provides fresh insight into the mechanism of HDR in humans, and its regulation by the BRCA2 tumor suppressor.

Fig. 1.

The electrophoretic mobility of RAD51 complexes on a resected dsDNA substrate is altered by BRCA2_[BRC1–8]. (A) Schematic representation of the protocol and reagents used for EMSA to detect association between BRCA2_[BRC1–8], RAD51, and resected dsDNA in the presence of RPA and ATP. (B) RAD51 forms complexes on a resected dsDNA substrate that are further retarded in an EMSA by increasing concentrations of BRCA2_[BRC1–8], suggesting co-complex formation. Radiolabeled resected dsDNA (10 μM) was premixed with 0.07 μM RPA before adding 3.3 μM RAD51 in the absence (lane 1) or presence of increasing concentrations of BRCA2_[BRC1–8] (0.4–1.6 μM; lanes 3–6) to reactions containing 1 mM ATP and incubated at 37 °C for 60 min. Protein-DNA complexes were resolved by gel electrophoresis and analyzed by phosphorimaging. In the presence of RAD51, the mobility of the resected dsDNA is resolved as a diffuse band (lane 2). The addition of increasing concentrations of BRCA2_[BRC1–8] further retards this RAD51-resected dsDNA complex (lanes 3–6). The mobility of the radiolabeled resected dsDNA alone is indicated in lane 1 as unbound (UB). Note that lanes 2–6 in this figure and in Fig. S2 are isolated from the same original gel, and lane 1 has been duplicated.

Fig. 2.

EM visualization of RAD51-ssDNA assemblies in the presence or absence of BRCA2_[BRC1–8]. RAD51 (5 μM) was incubated with 15 μM φX174 circular ssDNA either in the absence (A) or presence (B) of 2 μM BRCA2_[BRC1–8] in a reaction mix containing ATP for 15 min at 37 °C. After staining with 1% uranyl acetate, the grids were analyzed for protein-DNA complexes. In the absence of BRCA2_[BRC1–8], visualization by EM reveals few DNA-bound assemblies of RAD51, but a large number of oligomeric RAD51 rings (black arrows) apparently free of DNA (compare A1 with B1). BRCA2_[BRC1–8] markedly reduces the number of oligomeric RAD51 rings. This is accompanied by an increase in ssDNA-bound protein assemblies that appear as extensively aggregated masses. (Scale bar, 100 nm.) Quantitation (see SI Methods) to compare the ratios of areas occupied by rings, with areas enclosing filaments, revealed an 11-fold reduction.

Fig. 3.

Effect of BRCA2_[BRC1–8] on RAD51-ssDNA association measured by TCCD. (A) Single-molecule bursts from RAD51-Atto647N (red) and Alexa488-ss/dsDNA (blue). Coincident bursts in both channels indicate the presence of a RAD51-DNA complex diffusing through the probe volume. RAD51-Atto647N (3.3 μM) alone or in the presence of either 1.2 μM BRCA2_[BRC1–8] or 2 mM Ca²⁺ ions were incubated with 10 μM Alexa488-ssDNA to measure (B) the fraction of Alexa488-ssDNA fluorescent bursts that show a coincident RAD51-Atto647N burst (corrected for detector efficiency) and (C) the mean ratio of intensities observed in coincident RAD51-Atto647N and Alexa488-ssDNA bursts. Calcium ions have been shown to stabilize RAD51 filaments on DNA (25) and were used as a positive control in the TCCD experiments.

Fig. 4.

BRCA2_[BRC1–8] delays RAD51 assembly on dsDNA. (A) Schematic of the AflIII restriction enzyme digestion pattern of radiolabeled ApaLI-linearized ΦX174 dsDNA. (B) Radiolabeled dsDNA (7 μM) was incubated either in the absence (lane 1) or presence of increasing concentrations of RAD51 (0.5–4.0 μM; lanes 2–7) with AMP-PNP at 37 °C for 60 min. Half of the reaction was removed and mixed with gel loading buffer for EMSA, while the other half was treated with 10 U AflIII at 37 °C for 15 min before being deproteinized. The AflIII digestion pattern (Upper) was then directly compared to ternary complex formation (Lower). Migration of the ternary complex was progressively retarded as a function of RAD51 concentration and correlated with a decrease in the ability of AflIII to digest the dsDNA (compare lane 1 with lanes 2–7). Note that only the radiolabeled end-fragments are visible in lane 1 (828 and 2632 bp); the central fragment is unlabeled and can only be seen in a partial digest. The radiolabeled linear dsDNA alone (lane 1) is marked UB. (C) BRCA2_[BRC1–8] delays RAD51 assembly on dsDNA. Reactions were assembled and analyzed as above, except that ATP was used instead of AMP-PNP and BRCA2_[BRC1–8] was added at the indicated concentrations (lanes 5–16). Whereas 3.3 μM RAD51 assembles rapidly and completely on dsDNA, conferring protection from restriction digestion, the addition of 1–1.9 μM BRCA2_[BRC1–8] retards assembly in a concentration-dependent manner. Single-molecule fluorescence data, in which 3.3 μM RAD51-Atto647N alone or in the presence of either 1.2 μM BRCA2_[BRC1–8] or 2 mM Ca²⁺ ions were incubated with 10 μM Alexa488-dsDNA for 7.5 min (dark) and 15 min (light) to measure (D) the fraction of Alexa488-dsDNA fluorescent bursts that show a coincident RAD51-Atto647N burst (corrected for detector efficiency) and (E) the mean ratio of intensities observed in coincident RAD51-Atto647N and Alexa488-dsDNA bursts.

Fig. 5.

BRCA2_[BRC1–8] stimulates RAD51-mediated strand exchange while delaying RAD51-dsDNA assembly. Strand exchange reactions between 10 μM resected dsDNA and 7 μM homologous dsDNA mediated by 3.3 μM RAD51 were assembled in the presence or absence of BRCA2_[BRC1–8]. (A) The dsDNA substrate was radiolabeled to allow RAD51 assembly on dsDNA to be monitored via protection from AflIII digestion as described in Fig. 4. (B and C) The resected dsDNA substrate was labeled to allow the detection of joint molecule products of recombination. Without BRCA2_[BRC1–8] (A, lanes 1–4), rapid protection of the dsDNA by RAD51 is evident between 0–7.5 min; and joint molecule (jm) formation is not detectable (B, lanes 1–5). By contrast, the addition of 1–1.9 μM BRCA2_[BRC1–8] to the strand exchange reactions markedly delays RAD51 assembly on dsDNA (A, lanes 5–16). Delayed RAD51 assembly on dsDNA by 1.3 μM BRCA2_[BRC1–8] between 15–60 min (A, lanes 9–12) is accompanied by the stimulation of jm formation (B, lanes 6–10). (B) A DNA species co-purifying with the resected dsDNA substrate (Fig. S1B) is marked “ns,” whose relative intensity is unaltered between the lanes, indicating it does not interfere with the reaction. The bottom panel compares the formation of jm products in the presence or absence of BRCA2_[BRC1–8] as a percentage of the resected dsDNA substrate, with the intensity of the ns band expressed in arbitrary units. (C) Shows an extended time-course analysis for a strand-exchange reaction carried out as in B, in the presence of BRCA2_[BRC1–8]. (D) Shows the quantitation of jm formation from the data in B and C. Error bars indicate SEM.

Fig. 6.

Opposing effects of BRCA2_[BRC1–8] on RAD51 binding to ssDNA versus dsDNA together bolster one another to stimulate strand exchange. A hypothetical model for the regulation of RAD51-mediated HDR by BRCA2_[BRC1–8] is depicted. It refers to events that occur after the resection of a DSB to generate a 3′-tailed ssDNA substrate, but does not show the putative role of the C-terminal ssDNA-binding domain of BRCA2 in displacing RPA, and guiding RAD51 to its substrate near ssDNA/dsDNA junctions (9). When BRCA2_[BRC1–8] is absent (left-hand side), ssDNA and dsDNA compete for RAD51 binding. Strand exchange is inefficient because premature, stable RAD51 assembly on dsDNA is futile. BRCA2_[BRC1–8] favors the preferential assembly of RAD51 onto ssDNA because it promotes RAD51-ssDNA binding while suppressing RAD51-dsDNA binding (right-hand side). These opposing effects reinforce one another to guide stepwise progression from the presynaptic to the synaptic step of strand exchange.