Figure 1.

Identification of MERIT40 as a novel BRCA1–Rap80 complex constituent that is required for BRCA1 DSB localization. (A) Coomassie-stained gel representing eRap80 (1–719 and Δ233–399) complexes after consecutive Flag and HA affinity-purification steps from HeLa S3 nuclear extracts. The identities of both eRap80 species and the four Coomassie-stained bands that are uniquely present in the eRap80 wild-type complex are indicated as Abraxas, BRE/BRCC45, BRCC36, and C19orf62. (B) IF for endogenous MERIT40 in HeLa cells at 4 h following IR (top panel) and 30 min after laser microirradiation (bottom panel), demonstrating MERIT40 colocalization with γH2AX at DSBs. (C) Immunoblot (IB) of the eMERIT40 complex after Flag IP from HeLa S3 cells. BRCC36, Abraxas, Rap80, and BRCA1 were detected as MERIT40-associated proteins. (D) MERIT40 interacts with the BRCA1 BRCT domain, but not a clinical BRCT domain mutant. Nuclear extracts of HeLa S3 cells expressing eMERIT40 were incubated with recombinant GST, GST-BRCT, or GST-M1775R proteins bound to GST beads. (Top) IB with anti-HA antibody was performed to detect eMERIT40. (Bottom) The Ponceau-stained membrane is displayed showing the amount of each GST fusion protein. (E,F) MERIT40 is required for BRCA1 DSB localization. IF was performed for BRCA1 following Ct or MERIT40 siRNA treatment in IR-treated (E) and laser-microirradiated (F) HeLa cells. MERIT40 siRNA A and B target different sequences in the coding region of the MERIT40 mRNA. (G) Quantification of BRCA1 stripes as a percentage of 53BP1 stripes displayed graphically from F. At least 200 cells were counted in triplicate for the analysis. Error bars represent standard deviation (SD) and P values were calculated by the Student's t-test. (H) MERIT40 is required for BRCA1 association with Rap80. BRCA1 IP was performed in HeLa S3 cells following transfection with MERIT40 or Ct siRNA. IB was performed on the BRCA1 IP eluates as indicated.