Journal: eLife

Article Title: BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair

doi: 10.7554/elife.79183

Figure Lengend Snippet: Figure 5. BRC2-S1221P and BRC7-T1980I fail to stimulate RAD51-ssDNA complex formation. (A) Schematic of reaction to assay BRC repeat stimulation of RAD51-ssDNA complex formation by EMSA. RAD51 was pre-incubated with increasing amounts of BRC protein for 15 min then radiolabeled ssDNA (dT40) was added for 40 min. All reactions were incubated at 37 degrees and visualized on a 6% TAE polyacrylamide gel. (B) Autoradiograms of EMSA gels depicting increasing concentration of BRC proteins: BRC2, BRC2-S1221P, BRC7, BRC7-T1980I incubated with 10 nM RAD51 and 400 pM ssDNA (dT40*). Lane 1 is no protein control. Lane 2 is RAD51 alone. (C) Quantification of BRC-RAD51-ssDNA complexes calculated from gels shown in B. Error bars represent the S.D. for two biological independent experiments. (D) Schematic of biotinylated DNA pull-down assay. Purified BRC proteins were pre-incubated with increasing concentrations of purified RAD51 for 10 min. Biotinylated ssDNA (167-mer) was then added for 10 min to allow nucleoprotein filament formation and captured on magnetic streptavidin beads. The beads were then washed, eluted in sample buffer, analyzed by SDS-PAGE, and bound RAD51 was detected by western blotting using an anti-RAD51 antibody. (E) Western blot depicting RAD51 pulled down and eluted from biotin-DNA-BRC-RAD51 complexes. 0, 50, or 100 nM RAD51 was pre-incubated with 80 nM of BRC peptide, incubated with biotin-ssDNA, washed extensively, and eluted. Densitometric quantitation of RAD51 binding at 100 nM is indicated in the boxes below. (F) Schematic depicting single- molecule FRET (smFRET) assay with 30-nucleotide 3’-tail ssDNA. Addition of BRC7WT results in an increase in RAD51 binding leading to a transition from high FRET (DNA-only) to medium FRET (BRC7WT and RAD51-bound). Histograms display a shift (ΔEFRET = 11%) upon addition of 20 nM BRC7WT and 20 nM RAD51 that is not observed in BRC7T1980I or in the BRC2 peptides (WT and S1221P). Representative histograms do not include zero FRET values or photobleached portions of the FRET trajectories. A minimum of 250 smFRET trajectories were utilized to generate each histogram.

Article Snippet: Purified proteins were visualized on a 4–15% gradient SDS- PAGE TGX Stain- Free gel (Bio- Rad 456–8086) and then stained with Coomassie blue (Bio- Rad 1610786).

Techniques: Incubation, Concentration Assay, Control, Pull Down Assay, Purification, SDS Page, Western Blot, Quantitation Assay, Binding Assay, Smfret Assay