Fig. 3: Bre1 promotes Rad51-ssDNA filament assembly and strand exchange in vitro.

a, g Scheme showing the ssDNA pull-down assays. b, An ssDNA pull-down assay followed by Western blot analysis showing the effect of Bre1 on Rad51 binding to ssDNA. c Quantification of the relative amount of ssDNA-bound Rad51 presented in (b). Values represent the mean ± SD of three independent experiments (n = 3). d An electrophoretic mobility shift assay showing the effect of Bre1 or EK2A protein on Rad51 binding to M13 mp18 circular ssDNA. e A single-molecule magnetic twister system for monitoring the kinetics of Rad51 assembling on ssDNA. The ssDNA was stretched using MT at 8 pN, 20 ^oC, and pH 7.5. f Single-molecule MT analysis showing the effect of Bre1 or the peptide pep104 on Rad51 assembling with ssDNA. 150 nM of Rad51 and 150 nM of Bre1 or Pep104 were used for experiments. h, i An ssDNA pull-down assay and quantification showing the effect of Bre1 or EK2A on the replacement of RPA by Rad51. Values represent the mean ± SD of three independent experiments (n = 3). j, k Single-molecule MT analysis of the effect of Bre1 or Pep104 on the replacement of ssDNA-bound RPA by Rad51. ssDNA was initially saturated with 300 nM of RPA. Subsequently, 750 nM of Rad51 with or without Bre1(750 nM) or Pep104(750 nM) was used for the reaction. The average (dark-colored) and SD (lighter-colored) of the time courses at each condition in (f) and (k) were obtained from three independent molecules (n = 3). l Scheme showing the assay for measuring the strand exchange reaction. m An EMSA assay showing the effect of Bre1 on Rad51-mediated strand exchange. n Quantification of the relative amount of the strand exchange products showed in (m). Values represent 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 for panels are provided as a Source Data file.