Fig. 5: Bre1 physically counteracts Srs2 to facilitate Rad51 loading and HR repair.

a Immunoprecipitation(IP) followed by Western blot analysis showing the interaction between Srs2-3xFLAG and Bre1-3xHA. IgG was used as a mock IP. GAPDH serves as a loading control. b A GST pull-down assay indicating the interaction between 6xHis-Bre1 and GST-Srs2. GST was used as a control. The products were resolved on SDS-PAGE followed by Coomassie blue staining. c, f ChIP analysis of Srs2-3xFLAG or Rad51-3xFLAG loading at 1 kb away from the DSB 4 h after break induction. Values in (c) and (f) are the mean ± SEM of three independent experiments (n = 3). d Scheme showing the WT, truncated or mutated Bre1 protein used for GST pull-down assay. The Bre1 motifs are indicated by the colorful bars. e GST pull-down assay and Western blot analysis of the interaction between GST-Srs2 and 6xHis-tagged WT, truncated or mutated Bre1 protein. The amount of GST-Srs2 used for experiments is indicated by Coomassie blue staining (lower panel). The product was detected by Western blot with an anti-His antibody (upper panel). g The survival rate of DSB repair by ectopic recombination in indicated strains. Values are the mean ± SEM of three independent experiments (n = 3). h DNA damage sensitivity test for indicated strains. Drug concentrations are indicated. Statistical analysis was calculated with the Student t-test (two-tailed). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.