Figure 6. BRIT1 is required for recruitment of RAD51/BRCA2 to the IR-induced DNA damage sites.

(A) RAD51/BRCA2 foci formation was inhibited in IR-treated BRIT1 ^−/− MEFs. The WT and mutant MEFs on the coverslips were treated with or without ionizing radiation (5 Gy), then subjected to immunofluorescent staining 30 min later. IR-induced RAD51 foci were diminished in BRIT1 ^−/− MEFs (d) compared to those in WT (b). Similarly, compared to WT (j), BRCA2 foci in mutant MEFs (l) were barely formed in response to IR. Scale bar, 10 µm. (B) Chromatin-bound RAD51 and BRCA2 were dramatically reduced in IR-treated BRIT1 ^−/− MEFs. MEFs were treated with or without ionizing radiation (8Gy) and collected 1 h later. Chromatin pellets were then subjected to chromatin isolation and Western blot analysis, and probed with antibodies against BRIT1, RAD51, BRCA2, p-ATM, ATM or ORC2, respectively. ORC2 was used as a loading control. Chromatin-bound p-ATM or ATM remained the same between the WT and the mutant cells. However, there was much fewer RAD51 or BRCA2 bound to chromatin in the mutant MEFs as compared to the WT. p-ATM: phosphorylated ATM. (C) RAD51 or BRCA2 protein expression was not altered due to loss of BRIT1. Total protein lysates from indicated MEFs were used to detect the protein levels of RAD51/BRCA2. RAD51/BRCA2 expression was comparable between the WT and mutant MEFs. (D) BRIT1 physically associated with RAD51/BRCA2. The immortalized BRIT1 ^+/+ MEFs were transfected with vector or FLAG-BRIT1 and the cell lysates were collected 1 h after irradiation (8Gy), subjected to anti-FLAG immunoprecipitation assay, separated by SDS-PAGE, and blotted with anti-FLAG, anti-RAD51, or anti-BRCA2 antibodies, respectively.