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. 2006;34(14):4000-11.
doi: 10.1093/nar/gkl505. Epub 2006 Aug 16.

A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange

Mahmud K K Shivji  1 , Owen R Davies, Jane M Savill, Debbie L Bates, Luca Pellegrini, Ashok R Venkitaraman
Affiliations

A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange

Mahmud K K Shivji et al. Nucleic Acids Res. 2006.

Abstract

Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of approximately 30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2(BRC1-8)) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2(CTD)). BRCA2(BRC1-8) binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH4+ are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2(BRCI-8) stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2(CTD) through a previously unrecognized role in control of RAD51 activity.

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Figures

Figure 1
Figure 1
Expression and purification of a 1127 amino acid fragment of BRCA2 containing all 8 BRC repeats. (A) Human BRCA2 is a large protein of 3418 amino acids that can be divided into three general regions: an N-terminal region of unknown function, a central exon 11 region and a C-terminal DNA-binding domain (in addition to an extreme C-terminal RAD51-binding site encoded by exon 27). The eight RAD51-binding BRC repeats are located well spaced from one another amidst the long and divergent exon 11 region. We cloned and expressed a 1127 amino acid fragment of human BRCA2 corresponding to amino acid residues 987–2113 and spanning all 8 BRC repeats (BRCA2BRC1–8). (B) The 126 kDa BRCA2BRC1–8 protein was expressed in E.coli as an insoluble protein; it was purified under denaturing conditions by anionic exchange chromatography using Q Sepharose, and then cationic exchange chromatography using SP Sepharose. The resulting purified denatured sample was renatured in a buffer containing 500 mM l-arginine and was then soluble and stable after dialysis into a standard protein buffer. (C) Co-immunoprecipitation of purified BRCA2BRC1–8 with human RAD51; purified BRCA2 and/or purified RAD51 were incubated with pre-formed complexes of protein A–Sepharose and rabbit polyclonal anti-RAD51 antibodies or non-immune rabbit serum IgG. The resulting complexes were visualized by SDS–PAGE and protein staining with SimplyBlue, showing that the purified BRCA2BRC1–8 protein is functional in RAD51 binding. (D) An interaction between BRCA2BRC1–8 and RAD51 is further confirmed by co-immunoprecipitation anti-BRCA2 antibodies (visualized by western blot using anti-RAD51 antibodies). BRCA2BRC1–8 (10 nM) was incubated with 1–100 nM RAD51 before immunoprecipitation using pre-formed complexes of protein A–Sepharose and anti-BRCA2 or control irrelevant IgG. (E) The RAD51-binding capacity of BRCA2BRC1–8 was tested through an immunoprecipitation of BRCA2BRC1–8 (0.1 µM) with anti-BRCA2 antibodies, in the presence of 0.05, 0.1, 0.5 and 1.0 µM RAD51 (providing molar ratios of 0.5, 1, 5 and 10:1). Increasing amounts of RAD51 were co-immunoprecipitated at molar ratios between 0.5:1 and 5:1 (detected using anti-RAD51 antibodies), with binding saturation between 5:1 and 10:1. This suggests the presence of multiple RAD51-binding sites within each BRCA2BRC1–8 molecule.
Figure 2
Figure 2
Circular dichroism spectroscopy of purified BRCA2BRC1–8. (A) A far UV spectrum recorded over the range 185–260 nm is indicative of a mixture of secondary structural elements. Deconvolution of this spectrum using the CDSSTR algorithm indicates that the purified BRCA2BRC1–8 protein consists of 9% α-helix, 33% β-sheet, 24% turns and 34% random coil (data were fitted with normalized r.m.s.d. of 0.031). This mixture of structured regions and flexible unstructured sequence is consistent with the predicted nature of the exon 11 region of BRCA2 given its generally poor conservation in terms of sequence but strong conservation in terms of inter-BRC repeat spacing between mammalian BRCA2 orthologues. (B) The differences between far UV spectra recorded over the range 205–360 nm under native and denaturing (6 M guanidine–HCl) conditions confirm that the renatured BRCA2BRC1–8 sample does contain secondary structural elements that are destroyed upon denaturation.
Figure 3
Figure 3
EMSA analysis of ternary complex formation between BRCA2BRC1–8, RAD51 and DNA. The BRCA2BRC1–8 fragment induces a concentration-dependent shift in the mobility of RAD51/DNA complexes (lane 2) regardless of whether radiolabelled DNA is added to a pre-formed RAD51–BRCA2BRC1–8 complex (lanes 3–9), or BRCA2BRC1–8 is added to a pre-formed RAD51 filament on radiolabelled DNA (lanes 10–16). The dashed line marks the lower edge of RAD51–DNA complexes (marked B for bound). The first lane shows the migration of radiolabelled dsDNA without added protein (marked UB for unbound). Note that BRCA2BRC1–8 alone has no effect on the mobility of the radiolabelled dsDNA (lanes 17 and 18).
Figure 4
Figure 4
BRCA2BRC1–8 forms a ternary complex with RAD51 on DNA-joint molecules. (A) Schematic representation of the binding assay. Proteins were pre-incubated in strand exchange buffer containing 1 mM AMP-PNP (Materials and Methods) at 37°C for 30 min, before the addition to different concentrations of DNA-joint molecules bound to streptavidin-conjugated magnetic beads, and incubation for a further 30 min at 37°C. Proteins in the supernatant (s) were separated from the protein–DNA complexes bound to the magnetic beads (b) before analysis by SDS–PAGE. (B) Binding of BRCA2BRC1–8 is dependent on RAD51. SDS–PAGE analysis of proteins in the supernatant (s) or bound to DNA-joint molecules (b) is shown. DNA-joint molecules were incubated with RAD51 (5 µM) alone (lanes 1 and 2), BRCA2BRC1–8 (2 µM) alone (lanes 3 and 4) or RAD51 with BRCA2BRC1–8 (lanes 5 and 6). (C) Ternary complex formation between BRCA2BRC1–8 and RAD51 bound to DNA-joint molecules occurs at different ssDNA/RAD51 concentration ratios. Complex formation by RAD51 (5 µM) is shown at ssDNA/RAD51 ratios of 2:1 or 3:1 in the absence (lanes 1–4) or presence (lanes 5–8) of BRCA2BRC1–8 (2 µM). (D) Ternary complex formation by RAD51 (5 µM) and DNA-joint molecules at an ssDNA/RAD51 ratio of 3:1 with increasing concentrations of BRCA2BRC1–8. Lanes 1 and 5 represent control reactions performed in the absence of BRCA2BRC1–8. The (s) and (b) fractions are indicated.
Figure 5
Figure 5
RAD51-mediated strand exchange assay. (A) Schematic representation of the strand exchange assay. (B) Stimulation of RAD51-mediated strand exchange in the presence of (NH4)2SO4 requires RPA. Reaction mixtures containing 100 mM (NH4)2SO4 were pre-incubated without (lanes 1 and 5) or with increasing concentration of RAD51 (lanes 2–4 and 6–8) and ssDNA (30 µM) for 5 min followed by buffer control (lanes 1–4, no RPA) or with RPA (1 µM; lanes 5–8) for another 5 min. Linear dsDNA (30 µM) was added to stimulate strand exchange for a further 60 min. Reactions were performed at 37°C. The reactions were de-proteinized and analysed by gel electrophoresis and ethidium bromide staining. (C) Stimulation of RAD51-mediated strand exchange as function of ssDNA titration. Reactions were set up in the presence of (NH4)2SO4 and RPA as described in (B) except that the RAD51 concentration was constant at 10 µM (lanes 2–7) in the absence (lanes 1 and 2) or presence (lanes 3–7) of increasing concentration of ssDNA. Linear dsDNA was present at 10 µM. The reactions products were analysed as described previously. The ssDNA/RAD51 ratio is as indicated.
Figure 6
Figure 6
Stimulation of RAD51-mediated strand exchange by BRCA2BRC1–8. (A) Stimulation of RAD51-mediated strand exchange by BRCA2BRC1–8 in the presence of KCl and Mg2+. Reactions were carried out using ssDNA and RAD51 at a ratio of 3:1. RAD51 was pre-incubated without (lane 1) or with increasing concentrations of BRCA2BRC1–8 (lanes 2–4) for 5 min before adding ssDNA and RPA (0.26 µM). After a further 5 min incubation, radiolabelled linear dsDNA (10 µM) was added and incubated for a further 60 min. The reactions steps were incubated at 37°C. The strand exchange products were analysed by gel electophoresis and phosphoimager. (B) Stimulation of RAD51-mediated strand exchange by BRCA2BRC1–8 is dependent on Mg2+ and ATP. Reactions were carried out as described in (A) either in the absence of both co-factors Mg2+ and ATP (lanes 1–4), in the absence of ATP but presence of Mg2+ (lanes 5–8) or in the presence of both co-factors (lanes 9–12). BRCA2BRC1–8 was present as indicated. (C) Effect of BRCA2BRC1–8 (lane 2) or isolated BRC4 peptide (lane 3) on RAD51-mediated strand exchange when BRC repeats are present at equivalent molarities. Reactions were carried out as in (A). The first lane shows a reaction without added proteins. (D) The effect of BRCA2BRC1–8 on RAD51-mediated strand exchange is not dependent on physiologically relevant Ca2+ concentrations. Reactions are as described in (B) except that RAD51, either in the absence (lanes 1–5) or presence of BRCA2BRC1–8 (2 µM; lanes 6–10), was incubated with buffer containing no Ca2+ ions (lanes 1 and 6) or an increasing concentration of Ca2+ (lanes 2–5 and 7–10). (E) BRCA2BRC1–8 stimulates RAD51-mediated strand exchange at different DNA/RAD51 ratios. Reactions are as described in (A).
Figure 7
Figure 7
A model for the multiple roles of BRCA2 in regulating RAD51-mediated homologous recombination BRCA2 sequesters RAD51 in an inactive complex in undamaged cells. Following DNA damage, the activated BRCA2–RAD51 complex is targeted via the C-terminal domain (CTD) to the linear-duplex DNA of the RPA-coated, resected single-stranded ends. The CTD is essential to displace RPA, enabling the BRCA2–RAD51 complex to bind DNA to form the ternary complex demonstrated here (Figures 3 and 4). This may help to orient the BRCA2–RAD51 complex with respect to the polarity of the resected double-strand break. Our results (Figure 6) suggest that the multi-BRC repeat region of BRCA2 can stimulate RAD51-mediated strand exchange, distinct from the RPA-displacing activity of the CTD, a novel activity that promotes homologous recombination. We speculate that stimulation of strand exchange involves the same mode of RAD51–BRC repeat binding as in the inactive complex, with each BRC repeat capping one self-association surface of RAD51, but permitting filament growth from its opposite surface (circled inset). The resulting nucleoprotein filament might thus consist of multiple RAD51 multimers separated by interleaved BRC repeats.
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