(a) BRIT1-SWI/SNF interaction is enhanced in the presence of DNA damage signaling. Lysates were prepared from cells transfected with empty vector (V) or Flag-BRIT (B) at the indicated time points after exposure to IR (10 Gy) and immunoprecipitated. (b) DNA-damage induced BRIT1-SWI/SNF interaction is dependent on ATM/ATR signaling. Cells were harvested 15 minutes after exposure to IR (10 Gy). (c) BAF170 is a substrate candidate for ATM/ATR (Top and Middle) and its phosphorylation is dependent on the presence of ATM/ATR (Bottom). (d) The mutation on BAF170 (S969) suppressed the recognition of BAF170 by the p-S/TQ antibody after IR. A series of mutations on BAF170 were generated to replace potential ATM/ATR target sites S/TQ to AQ. Chk2-pulldown by p-S/TQ was used as a positive control to show that the general binding activity of p-S/TQ to other ATM/ATR substrates was not affected in cells transfected with M7 mutant. (e) BAF170 (S969) is phosphorylated in vitro by ATM/ATR kinase assay. The sequence around BAF170 (S969) was cloned into GST vector and the phospho-mutant (S969A) was made in this vector.

(a) Impaired chromatin relaxation analyzed by MNase sensitivity assay. Cells were exposed to 4 Gy IR. (Top) Representative image. (Bottom) Quantification of average nucleosome size. Each value represents the mean ± SEM of of three independent experiments; Student’s t-test. (b) The function of BRIT1 in DNA repair is dependent on its interaction with SWI/SNF. BRIT1-deficient cells were reconstituted with indicated plasmids. BRIT1 with N-terminal deletion (BRIT1-ND), which lacks its SWI/SNF interaction domain, was unable to rescue the defect of DNA repair to a similar extent to the BRIT1 BRCT-Δ3 mutant that lost its activity to be recruited to DNA damage lesions. Each value is relative to the percentage of GFP+ cells in I-SceI-transfected cells with control siRNA transfection, which was set to 1 and represents the mean ± SD of three independent experiments. (c) Requirement of BRIT1 for Rad51 foci formation. (Top) Representative immunostaining images. Scale bar is 10 µM. (Bottom) The bar graph represents the mean ± SD of three independent experiments; Student’s t-test. At least 50 cells were scored in each sample. (d) Restored HR repair in BRIT1 deficient cells in the presence of chromatin relaxation agents. Each value is relative to the percentage of GFP+ cells without siRNA transfection, which was set as 1, and represents the mean ± SD of three independent experiments. Western blot analyses to demonstrate the effective BRIT1 knockdown were shown next to the graph.

(a) Silver staining of the BRIT1 complex separated by SDS-PAGE. The whole cell extracts were prepared from U2OS cells transiently transfected with empty vector or Flag-BRIT1. Subunits of SWI/SNF are indicated. (b) Co-IP of SWI/SNF with BRIT1 analyzed by Western blotting from cells transfected with empty vector (V) or Flag-BRIT1 (B). (c) BRIT1-SWI/SNF interaction dependent on BAF170 (Left) and BAF155 (Right). (d) N-terminal BRIT1 is required for BRIT1-SWI/SNF interaction. (Top) Schematic diagram of BRIT1 deletions. (Bottom) Co-immunoprecipitation of BAF170 with Flag-BRIT1 containing indicated deletions. (e) SWI/SNF interacts specifically with the N-terminal BRIT1 in vitro. Lysates of 293T cells were treated with or without lambda phosphatase, and incubated with purified GST or GST-N-terminal BRIT1. MCM-2 phosphorylation (S40/41) was used as a positive control for effective phosphatase treatment.

(a) Defective HR repair in BRIT1-depleted-cells transfected with BRIT1 siRNA #1 (#1) and siRNA #2 (#2) upon DSB induced by I-SceI. (Left) Representative flow cytometry profile. (Right) Quantitative summary of at least three independent experiments. Each value is relative to the percentage of GFP+ cells in I-SceI-transfected cells without siRNA transfection, which was set to 1 and represents the mean ± SD of three independent experiments; Student’s t-test. (b) Defective NHEJ repair in BRIT1-depleted-cells transfected with BRIT1 siRNA #1 (#1) and siRNA #3 (#3) upon DSB induced by I-SceI. (Left) Representative image of PCR products digested by I-SceI or I-SceI+BcgI. (Right) Quantification of NHEJ repair product (uncut by I-SceI+BcgI). Percentage of uncut products in cells without siRNA transfection was set as 1. Each value represents the mean ± SD of three independent experiments; Student’s t-test. Western blot analyses to demonstrate the effective BRIT1 knockdown were shown next to the graph. (c) Impaired recruitment of SWI/SNF and DNA repair proteins to chromatin in BRIT1-depleted cells (Time course study and densitometry analyses were shown in supplementary Fig. 4a, b). (d) Impaired recruitment of SWI/SNF to the DNA damage site. ChIP analyses were performed at the indicated time points after induction of DSB by I-SceI transfection.

(a) Impaired DNA repair analyzed by comet assay after exposure of cells to IR. Percentage of cells with intact DNA (tail moment less than 2) in cells without IR exposure was set as 1. At least 100 cells were scored in each sample and each value represents the mean ± SEM of three independent experiments; Student’s t-test. Representative images are shown in Supplementary Fig. 7b. (b) Impaired cell survival in MCPH #1 cells exposed to DSB-inducing agents. Cells were pre-treated with the DNA replication inhibitor aphidicolin (Aphi) where indicated, and then treated with etoposide (Left) or camptothecin (Right). The graphs represent the mean ± SD of three independent experiments (c) Requirement of BRIT1 for foci formation of DNA repair proteins. (Top) Immunostaining of p-RPA foci. Scale bar is 10 µM. (Bottom) Quantification of p-RPA and Rad51 foci formation from three independent experiments (mean ± SD). At least 50 cells were scored in each sample for each experiment. (d) Reduced association of SWI/SNF to chromatin. (Top) Representative Western blots. (Bottom) Densitometry analyses of indicated protein values normalized against ORC2. Each value represents the mean ± SD of three independent experiments; Student’s t-test. (e) Impaired chromatin relaxation analyzed by MNase sensitivity assay. Cells were exposed to the DSB inducing agent neocarzinostatin (400 ng/mL). (Left) Representative image. (Right) Quantification of average nucleosome size. Each value represents the mean ± SEM of three independent experiments; Student’s t-test.