Figure 4. Regulation of HR by binding of Bag101 to Rad22 at the BAG domain.

(a) Illustration of Bag101 domains and the deletion mutants. (b) Physical interaction of Rad22 with Bag101 through the BAG domain. Immunoprecipitation was performed with FH6-tagged Bag101 for each cell containing a deleted domain; and immunoblotting was performed with anti-FLAG and anti-GFP antibodies to detect interactions. (c) HR frequency in each deletion mutant. Spontaneous HR frequencies were measured with the RDUX200 reporter gene. These experiments were performed three times and error bars represent the S.D. (d) Rad22-foci in deletion mutants. Cells were collected to count the number of Rad22-focus positive cells. Error bars represent the S.D. (e) The amount of Rad22 in Bag101 domain-deletion mutants. Rad22 levels in each mutant lacking the BAG or UBL domain were measured with Western blots using anti-Rad22 antibodies. (f) Dissociation of Rad22 from Bag101 after IR exposures. Cells were irradiated with 500 Gy and at the indicated times after irradiation, were harvested for immunoprecipitation with a HATA tag. Cell lysates and immunoprecipitates were immunoblotted with anti-Rad22 and anti-HA antibodies. The intensities of the Rad22 band were calibrated with a value of 1.0 with 0 Gy.