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. 2010 Oct;17(10):1263-5.
doi: 10.1038/nsmb.1905. Epub 2010 Aug 22.

The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA

Tina Thorslund  1 , Michael J McIlwraith, Sarah A Compton, Sergey Lekomtsev, Mark Petronczki, Jack D Griffith, Stephen C West
Affiliations

The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA

Tina Thorslund et al. Nat Struct Mol Biol. 2010 Oct.

Abstract

Individuals with BRCA2 mutations are predisposed to breast cancers owing to genome instability. To determine the functions of BRCA2, the human protein was purified. It was found to bind selectively to single-stranded DNA (ssDNA), and to ssDNA in tailed duplexes and replication fork structures. Monomeric and dimeric forms of BRCA2 were observed by EM. BRCA2 directed the binding of RAD51 recombinase to ssDNA, reduced the binding of RAD51 to duplex DNA and stimulated RAD51-mediated DNA strand exchange. These observations provide a molecular basis for the role of BRCA2 in the maintenance of genome stability.

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Figures

Figure 1
Figure 1
Purified BRCA2 binds specifically to ssDNA. (a) N- or C-terminally FLAG-tagged BRCA2 was purified from human cells and analyzed by SDS-PAGE and SyPro Ruby staining. M, size marker. (b) Purified N-terminally Flag-tagged BRCA2 was incubated with 5′-32P end-labeled ssDNA or dsDNA, and complexes were analyzed by agarose gel electrophoresis and visualized by autoradiography. (c) Electron microscopic visualization of BRCA2. Top: large field of N-terminally Flag-tagged BRCA2 protein, rotary shadowcast with tungsten. Scale bar, 40 nm. Arrows, ball-shaped BRCA2 particles; arrows with asterisks, rod-shaped particles. Below: individual protein particles at higher magnification. Scale bar, 25 nm. Images are shown in reverse contrast.
Figure 2
Figure 2
Binding of BRCA2 to tailed duplex DNA. N-terminally Flag-tagged BRCA2 protein was incubated with linear duplex DNA (3.5 kb long) containing a 54-nt ssDNA overhang at one end. Samples were fixed, mounted on glow-discharged carbon grids and rotary shadowcast with tungsten. Top: BRCA2 localizes to ssDNA tails of the duplex DNA substrate. Scale bar, 100 nm. Bottom: higher magnification images of dumbbell-shaped BRCA2 dimers bound to ssDNA tails. Scale bar, 50 nm. Images are shown in reverse contrast.
Figure 3
Figure 3
BRCA2 targets RAD51 to ssDNA and stimulates DNA strand exchange. (a) BRCA2 and RAD51 were mixed in binding buffer for 5 min at 24 °C, before the addition of 32P-labeled ssDNA or dsDNA. Incubation was then continued for 10 min, before complexes were fixed and analyzed by agarose gel electrophoresis and autoradiography. (b) BRCA2 was incubated for 5 min at 24 °C with 32P-labeled ssDNA, before RAD51 addition. Incubation was then continued for a further 5 min. Complexes were analyzed as in a. (c) Stimulation of RAD51-mediated strand exchange by BRCA2. Tailed duplex DNA was incubated with BRCA2 for 5 min before the addition of RAD51. After a further 5 min, 5′-32P end-labeled (*) duplex DNA (35 bp) was added and incubation continued for 10 min. The products were deproteinized and analyzed by 10% PAGE, followed by autoradiography.
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