RAD51C is a nuclear protein and forms foci following DNA damage. (A) Cell-free extracts were prepared from mock-treated DR95 cells or DR95 cells treated with etoposide and fractionated into cytosolic (Cyt) and nuclear (Nuc) fractions. These fractions (25 μg) were analyzed for the presence of RAD51C. RAD51, γ-H2AX and GAPDH were used as controls for the cytoplasmic and nuclear fraction by Western blotting. (B) Localization of GFP-RAD51C in DR95 cells. (C) RAD51C foci formation following etoposide treatment (50 μM, 1 h). Immunofluorescence of RAD51C, γ-H2AX and merge pictures are depicted.

Knockdown of RAD51C affects RAD51 foci formation in human cells. Immunofluorescence was performed on DR95 cells treated with 600 nM mitomycin C (A) or cells pretreated with RAD51C siRNA followed by DNA damage with mitomycin C (B). DNA staining by DAPI (blue) and immunofluorescence of RAD51 (green), γ-H2AX (red) and merge pictures are depicted.

RAD51 foci formation in DR95 cells after DNA replication. Confocal micrographs depict DNA stained with DAPI (blue), RAD51 (green) and γ-H2AX (red). The merge picture is an overlay of the three channels.

(A) Interactions between purified human RAD51 and RAD51C-XRCC3 or RAD51B-C-D-XRCC2 and between RAD51 and RAD51C in vivo. Human RAD51, or a mixture with CX3 or BCX2, were immunoprecipitated with preimmune serum or the anti-hRAD51 as indicated, and visualized by Western blotting using anti-hRAD51 mAb 14B4 and anti-RAD51C mAb 2H11. Lane 1, purified RAD51C_his10 and RAD51. CX3 and RAD51 (lane 2) or BCDX2 and RAD51 (lane 3) were immunoprecipitated with RAD51 preimmune antibody. As controls, RAD51 (lane 4), CX3 (lane 5) or BCDX2 (lane 6) were immunoprecipitated with anti-hRAD51 pAb. CX3 and RAD51 (lanes 7–10) or BCDX2 and RAD51 (lanes 11–14) were immunoprecipitated with anti-hRAD51 pAb and complexes were washed with buffer containing NaCl, as indicated. (B) Direct interaction between RAD51 and RAD51C. SF9 cells were infected with RAD51 baculovirus (lane 2) or a mixture of RAD51 and RAD51C (lane 3) and immunoprecipitated with the anti-hRAD51C. Complexes were washed in lysis buffer containing 1 M NaCl and visualized by Western blotting using anti-hRAD51 mAb 14B4. Lane 1, purified human RAD51. (C) Expression of YFP-RAD51C in HEK293 cells. Lane 1, purified RAD51C_his10; lane 2, cells transfected with the empty vector pEYFP-N1; lane 3, cells transfected with pEYFP-N1-51C encoding a fusion between YFP and RAD51C. RAD51C was visualized using the anti-RAD51C mAb 2H11. (D) Expression of CFP-RAD51 in HEK293 cells. Lane 1, purified RAD51; lane 2, cells transfected with the empty vector pECFP-N1; lane 3, cells transfected with pECFP-N1-51 encoding a fusion between CFP and RAD51. RAD51 was visualized using the anti-RAD51 mAb 14B4. (E) Co-immunoprecipitation of endogenous RAD51 with YFP-RAD51C. Extracts from HEK293 cells transfected with pEYFP-N1-51C were prepared and protein complexes were precipitated using control IgG (lane 2) or anti-RAD51 pAb (lane 3), and visualized by Western blotting with anti-RAD51C and RAD51, as indicated. Lane 1: marker protein (RAD51C_his10). (F) Co-immunoprecipitation of endogenous RAD51C with CFP-RAD51. Extracts from HEK293 cells transfected with pECFP-N1-51 were prepared and protein complexes were precipitated using control IgG (lane 2) or anti-RAD51 pAb (lane 3) and visualized by Western blotting with anti-RAD51 and RAD51C, as indicated. Lane 1: marker protein (RAD51).

Knockdown of RAD51C and XRCC3 affects cellular proliferation. (A) Small interfering RNA inhibition of RAD51C. Lane 1, purified RAD51C_his10. Whole-cell extracts from mock-transfected DR95 cells (lane 2) or DR95 cells transfected with RAD51C siRNA (lane 3) were subjected to Western blotting with RAD51C or GAPDH antibodies (as a loading control). (B) Inhibition of RAD51C by siRNA destabilizes XRCC3. Whole-cell extracts from mock-transfected DR95 cells (lane 2) or DR95 cells transfected with RAD51C siRNA (lane 3) were subjected to Western blotting using anti-RAD51B, anti-RAD51D, anti-XRCC2 and anti-XRCC3. Lane 1, purified RAD51B_his10, RAD51D, XRCC2, XRCC3_his6. (C) RAD51C siRNA can knockdown wild-type RAD51C but not an siRNA-resistant form. Lane 1, purified RAD51C_his10. Whole-cell extracts from cells transfected with pcDNA3 (lane 2), pcDNA-51C (lane 3) or the siRNA-resistant RAD51C construct pcDNA-51C-Res (lane 4), RAD51C siRNA-treated cells transfected with pcDNA3 (lane 5), pcDNA-51C (lane 6) or pcDNA-51C-Res (lane 7) were subjected to Western blotting with RAD51C or GAPDH antibodies. (D) RNAi inhibition of XRCC3. Lane 1, purified XRCC3_his6. Whole-cell extracts from mock-transfected DR95 cells (lane 2) or DR95 cells transfected twice with XRCC3 siRNA (lane 3) were subjected to Western blotting with XRCC3 or GAPDH antibodies. (E) Cellular proliferation of mock-treated or cells transfected once (top panel) or twice with an siRNA specific for RAD51C (middle panel). Bottom panel: wild-type or RAD51C siRNA-transfected cells challenged with 50 μM etoposide for 1 h followed by growth and recovery in fresh media. (F) Quantification of colony-forming assays. Cells were either untreated or treated with 50 μM etoposide for 1 h followed by growth and recovery in fresh media, as indicated. White bars indicate cells transfected once with RAD51C siRNA and gray bars designate cells transfected twice with RAD51C or XRCC3 siRNA. Black bars indicate wild-type cells. These experiments were repeated three times.

Foci formation of RAD51, RAD51C and 53BP1 on a unique DSB in vivo. DR95 cells were transfected with pCBASce and immunofluorescence was conducted with the indicated antibodies. Confocal micrographs depict DNA stained with DAPI (blue); RAD51 (top panel), anti-RAD51C (middle panel) and 53BP1 (bottom panel) (green); γ-H2AX (red). The merge picture is an overlay of the three channels.

Detection of repair proteins on a unique DSB in vivo at a high resolution by ChIPs. (A) RAD51C protein levels do not fluctuate during the cell cycle. Cells were synchronized, whole-cell extracts were prepared and subjected to Western blot analysis using RAD51C 2H11 mAb. The percentage of cells at each phase is represented. (B) Schematic representation of the position of primers used for real-time PCR quantification of ChIPs respective to the unique DSB created by I-SceI in vivo. (C–E) ChIPs of endogenous RAD51, RAD51C, Ku80 and MRE11 on a unique DSB. The presence of the proteins was verified during the G1 (C) or S–G2 phase (D) of the cell cycle. Real-time PCR on ChIP samples were carried out at 94–378, 675–1044 and 901–1210 nucleotides from the break (red, yellow and blue bars, respectively). (E) ChIP analysis of RAD51D, XRCC2 and XRCC3. Fold-enrichment represents the enrichment of the proteins compared to an IgG control (normalized with a PCR internal control to a locus other than the DSB). These experiments were repeated three times and the PCR reactions were performed in triplicate.