Fig. 4: Bre1 protects Rad51 filament from Srs2 dismantling activities via displacing Srs2 from ssDNA.

a Scheme showing an ssDNA pull-down assay. 200 nM of biotin-labeled ssDNA(90 nt) was immobilized to streptavidin-coated beads by incubating at room temperate for 30 min. Proteins were added in the indicated order. b An ssDNA pull-down assay followed by Western blot analysis indicating the role of Bre1 in antagonizing the effect of Srs2 on Rad51 filament. Protein and ssDNA concentrations are indicated. c Quantification of the relative amount of ssDNA-bound Rad51 showed in (b). Values are the mean ± SD of three independent experiments (n = 3). d An EMSA assay showing the role of Bre1 or Srs2 in Rad51-mediated strand exchange reaction. Protein concentrations are indicated. e Quantification of the relative amount of strand exchange products presented in (d). Values are the mean ± SD of three independent experiments (n = 3). f, g Scheme and EMSA showing the effect of Bre1 on the binding of Srs2 on ssDNA (M13 mp18, 100 nM). Proteins at indicated concentrations were added following the order indicated in (f). h ssDNA pull-down followed by Western blot analysis showing the effect of Bre1 on the binding of Srs2 to ssDNA (90 nt, 200 nM). i Quantification of the relative amount of ssDNA-bound Srs2 showed in (h). Values are the mean ± SD of three independent experiments (n = 3). Statistical analysis was calculated with the Student t-test (two-tailed). *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.