BCCIP interactions with BRCA2 and RAD51. (A) BCCIPα and BCCIPβ share a 258-N-terminal-amino-acid region including an acidic domain and a central conserved domain. Alternative mRNA splicing produces distinct C termini. BCCIP fragments used in immunoprecipitation assays (B) are shown below along with a summary of those results. (B) Mapping the BCCIP domain that interacts with BRCA2. Myc-tagged BCCIP fragments were coexpressed with a HA-tagged fragment of BRCA2 (aa 2883 to 3194) shown previously to interact with full-length BCCIPα in HeLa cells (30). Myc-tagged proteins were precipitated with mouse anti-Myc antibody and detected by rabbit anti-Myc antibodies in a Western blot (top panels). Coprecipitated HA-tagged BRCA2 fragment was detected by rabbit anti-HA antibody (bottom panels). (C) BCCIPβ interacts with BRCA2. Myc-tagged UBC9, BCCIPα, BCCIPβ, RAD51, and RAD52 were expressed in HeLa cells. Myc-tagged proteins were detected by Western blotting in whole-cell extracts or after precipitation with anti-Myc antibodies (top panel). Coprecipitated endogenous BRCA2 protein (bottom panel) was detected with anti-BRCA2 antibodies. Note that BCCIPα and BCCIPβ coprecipitate BRCA2 with approximately the same affinity. (D) Endogenous BCCIPα and BCCIPβ coprecipitate with endogenous BRCA2. Agarose-conjugated anti-BRCA2 beads were used to precipitate endogenous BRCA2 (top panel), and coprecipitated BCCIPα and BCCIPβ were detected by anti-BCCIP blotting (bottom panel). Nonspecific immunoglobulin G (IgG)-agarose beads were used as negative controls. WCE, whole-cell extract; IP, immunoprecipitation. (E) BCCIPα and BCCIPβ coprecipitate RAD51. Myc-tagged BCCIPα, BCCIPβ, and RAD52 were coexpressed with HA-tagged RAD51 in HeLa cells and precipitated with anti-Myc antibodies (top panels). Coprecipitated HA-RAD51 and endogenous RAD51 were detected by anti-RAD51 blotting (bottom panels), indicating that RAD51 is in complex with BCICPα and BCCIPβ.

BCCIP colocalizes with BRCA2 and RAD51. (A) Colocalization of BCCIP and BRCA2 in HT1080 cells. Chromatin-bound endogenous BCCIP was stained with anti-BCCIP (Texas Red, red) and BRCA2 was stained with anti-BRCA2 (FITC, green) in untreated cells or 8 h after an 8-Gy dose of gamma rays. Yellow foci in the merged images indicate colocalization. (B) Colocalization of BCCIP and RAD51. Cells were treated, and BCCIP was stained as described for panel A. RAD51 was stained with FITC. (C) Percentage of HT1080 cells with BCCIP foci colocalized with RAD51 or BRCA2 foci in cells treated with 0, 2, or 8 Gy of gamma rays and scored 8 h later. Cells with at least three colocalized BCCIP/BRCA2 or BCCIP/RAD51 foci were counted. (D) Percentage of BCCIP focus-positive (BCCIP⁺) cells with at least three colocalized RAD51 or BRCA2 foci in cells treated with 0, 2, or 8 Gy of gamma rays and scored 8 h later. (E, F) Percentages of BRCA2 focus-positive (BRCA2⁺) or RAD51 focus-positive (RAD51⁺) cells with colocalized foci. Cells were treated as described for panel D.

BCCIP is important for BRCA2 focus formation. (A) Entire populations of HT1080 cells were infected with adenoviruses expressing shRNAs targeted to BCCIPα, BCCIPβ, of both isoforms or with an empty vector as a control and stained with anti-BRCA2 antibodies (red) or DAPI to identify nuclei. Images were recorded 8 h after irradiation. (B) BCCIP expression in adenovirus-infected cells. Seventy-two hours after infection with adenoviruses expressing BCCIP-shRNAs, BCCIP expression was detected by anti-BCCIP blotting (top panel); anti-β-actin was used as loading control (bottom panel). (C) Average number of BRCA2 foci (± standard error) in cells with normal or reduced levels of BCCIPα or BCCIPβ. For each data point, foci were scored in ∼300 cells. BCCIP downregulation reduced BRCA2 foci significantly with or without irradiation, and irradiation increased BRCA2 foci significantly with or without BCCIP downregulation (all P < 0.0001). (D) Cumulative percentage of cells with at least a specified number of BRCA2 foci (x axis) in control and BCCIP-downregulated cells. BRCA2 foci were scored in unirradiated cells or 8 h after irradiation.

BCCIP is important for RAD51 focus formation. (A) About50% of HT1080 cell populations were infected with adenoviruses expressing GFP and shRNAs targeted to BCCIPα, BCCIPβ, or both isoforms or GFP alone as a control. Cells were irradiated with 8 Gy of gamma rays and prepared for immunofluorescence 8 h later. Cells were stained with anti-BCCIP antibodies (red); infected cells show green fluorescence. Within each field are infected and noninfected cells. Cells infected with BCCIP shRNA vectors (bottom three rows) show reduced BCCIP levels. (B) Cells from panel A were stained with anti-RAD51 antibodies (red). Infected (green fluorescence) and noninfected cells are shown in each panel. Cells infected with viruses expressing BCCIP shRNAs show reduced RAD51 foci. (C) Average number of RAD51 foci (± standard error) in cells with normal or reduced levels of BCCIPα or BCCIPβ (uninfected or adenovirus infected, respectively), 2 and 8 h after treatment with 8 Gy of gamma rays. For each data point, foci were scored in at least 300 cells. RAD51 foci are reduced only in cells infected with adenoviruses expressing BCCIP shRNAs. (D) Cumulative percentage of cells with at least a specified number of RAD51 foci (x axis) in control and BCCIP-downregulated cells. RAD51 foci were scored in uninfected or adenovirus-infected cells 2 and 8 h after treatment with 8 Gy of gamma rays.

BRCA2 is important for formation of BCCIP and RAD51 foci. (A) HT1080 cells were infected with adenovirus expressing shRNA targeted to BRCA2, and 72 h later, BRCA2 was detected by Western blotting. Empty virus was used as control; blots were also hybridized with anti-β-actin antibody as a loading control. (B) Average number of BCCIP foci (± standard error) in cells expressing normal or low levels of BRCA2, either unirradiated or irradiated with 8 Gy of gamma rays; foci were scored 8 h after irradiation. (C) Distribution of BCCIP foci in unirradiated or irradiated cells expressing normal or low levels of BRCA2. These data are plotted as described for Fig. 3D. (D, E) Average number and distribution of RAD51 foci in irradiated cells expressing normal or low levels of BRCA2. Data are presented as described for panels B and C, respectively.

BCCIP is critical for HRR. (A) Structure of HR substrate in HT256 cells. (B) Downregulation of BCCIP in HT256 cells. Cells were stably transfected with vectors expressing shRNAs targeted to BCCIPα, BCCIPβ, or both isoforms or with an empty vector as a control. Expression of both isoforms was monitored in two independent transfectants by Western blotting with anti-BCCIP antibodies (top panel). β-Actin serves as a loading control (middle panel), and expression of HA-tagged I-SceI nuclease was detected by anti-HA blotting (bottom panel). (C) HRR frequencies calculated as the number of G418-resistant colonies per viable cell plated in selective medium. HT256 cells expressed normal (control) or reduced levels of BCCIPα, BCCIPβ, or both isoforms. Values are averages ± standard errors for 4 determinations per cell line.