Figure 4: CCAR2 inhibits CtIP-mediated resection.

(a) Cells transduced with shRNAs against the indicated genes were irradiated (10 Gy). One hour after irradiation, cells were fixed and immunostained as indicated in the Methods section. The number of cells that show RPA foci was scored and represented as a percentage of the total. The graph represents the average and s.d.’s of three independent experiments. Representative images are shown at the right. Scale bar, 20 μm (b). The length of resected DNA was calculated using the SMART technique at individual DNA molecules. A Mann–Whitney test was performed to analyse the differences in dispersion. A representative experiment is shown. The median is shown in red. (c) The median of the resected DNA lengths was normalized to controls in cells depleted of the indicated proteins. The plot represents the average and s.d. of the normalized medians of four independent experiments. (d) DiVA cells were treated with 4-OHT to induce translocation of the nuclease AsiSI to the nucleus or were mock-treated, as described in the Methods section. Chromatin bound to RPA was immunoprecipitated and the occupancy of RPA was detected by qRT–PCR at 80 bp (left) or 800 bp (right) of three DSBs. DSB-3 represents a chromosome break that is exclusively repaired by NHEJ, whereas both NHEJ and HR can repair DSB-III and DSB-V. The same approach was performed in cells depleted for CCAR2 (black bars) or control cells (white bars). (e) Same as d, but using an antibody against CtIP for ChIP. (f) SMART assay with cells expressing the indicated plasmids and transfected with siRNA against CCAR2 (black bars) or a control sequence (siNT, white bars). Further details are as in c.