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Purified Human BRCA2 Stimulates RAD51-mediated Recombination


Purified Human BRCA2 Stimulates RAD51-mediated Recombination

Ryan B Jensen et al. Nature.


Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer.


Figure 1
Figure 1. Protein interactions with purified full-length human BRCA2
(a) Lane 1) 293T cell lysate, 2) amylose eluate, 3) purified BRCA2, 4) Western blot (Ab-2). “M”, standards. Asterisk, β-tubulin. (b) MMC survival. Wild type V79 (formula image), brca2−/− + MBP-BRCA2 (formula image), 2XMBP-BRCA2 (formula image), and brca2−/− VC8 (formula image) cells. Errors bars, s.d. (n=3). (c) and (d) Protein pull-downs. 2XMBP-BRCA2 with indicated proteins. “Marker”, protein input. ”Control”, proteins + amylose resin. (e) RAD51 titration of BRCA2: Lanes 1–4, RAD51 standards; 5–11, Pull-downs; 5, BRCA2 alone; 11, RAD51 alone; 12–14, BRCA2 standards. (f) Data from (e) fit to segmental linear regression. Errors bars, s.d. (n=2).
Figure 2
Figure 2. BRCA2 displays a strong preference for binding tailed and ssDNA substrates over dsDNA
(a) EMSA. BRCA2 binding: ssDNA, 3’ Tailed DNA, 5’ Tailed DNA, and dsDNA. (b) Quantification: 3’ Tailed DNA (formula image), 5’ Tailed DNA (formula image), ssDNA (formula image), and dsDNA (formula image). (c) EMSA in 0.5 M or 1 M NaCl: 3’ tailed (squares) or ssDNA (circles). Error bars, s.d. (n=3).
Figure 3
Figure 3. BRCA2 stimulates DNA strand exchange promoted by RAD51
(a) DNA strand exchange reaction protocol for (b) and (c). (b) Reaction in the absence (left) or presence (right) of RPA. (c) Quantification: absence (formula image); presence (formula image) of RPA. Error bars, s.d. (n=3). (d) DNA strand exchange protocol for (e) in absence of RPA. (e) Quantification of (d).
Figure 4
Figure 4. BRCA2 stimulates RAD51-mediated DNA strand exchange by promoting stable RAD51-ssDNA filament formation, overcoming inhibition by RPA
(a) DNA strand exchange reaction protocol for (b) – (c). (b) Autoradiograms: 3’ Tailed, 5’ Tailed, and ssDNA substrates. (c) Quantification of (b). (d) Assays as in (a), except RecA replaced RAD51 (red squares) and SSB replaced RPA (blue circles). (e) Inhibition of RAD51 ssDNA-dependent ATP hydrolysis by BRCA2. Error bars, s.d. (n=3).
Figure 5
Figure 5. BRCA2 does not anneal ssDNA complexed with RPA
(a) Schematic of DNA annealing assays. (b) Autoradiogram: absence of RPA, plus indicated protein (left); presence of RPA first, plus indicated protein (right). Lanes 1 and 14, radio-labeled 40-mer. (c) Quantification of (b): No Protein (grey); BRCA2 (blue); RAD52 (green); RPA (orange); RPA plus BRCA2 (yellow); RPA plus RAD52 (purple). Error bars, s.d. (n=3).

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