In vitro complex formation of Rad51–Rad52–ssDNA. (A) Schematic diagram of the assay. (B) Rad52 protein (1 μM) was incubated with a reaction mixture containing 3 μM (nucleotide) biotinylated-dT60 for 5 min, followed by the addition of various concentrations of Rad51 protein. After a 10-min incubation, the ssDNA complexes were purified and further processed as described in . One-tenth of the eluates were analyzed with Western blotting. Upper panel, Coomassie staining; lower panel, Western blotting using anti-Rad51 antibody. Lanes 1–4, in the absence of Rad52; lanes 5–8, in the presence of 1 μM Rad52. Lanes 1 and 5, no Rad51; lanes 2 and 6, 0.25 μM Rad51; lanes 3 and 7, 0.5 μM Rad51; lanes 4 and 8, 1.0 μM Rad51. (C) Various concentrations of Rad52 were incubated with 3 μM biotinated-dT60 for 5 min, followed by the addition of 1 μM Rad51. After a 10-min incubation, the mixtures were analyzed as described in . Upper panel, Coomassie staining; lower panel, Western blotting. Lanes 1–4, in the presence of 1 μM Rad51; lanes 5–8, in the absence of Rad51. Lanes 1 and 5, no Rad52; lanes 2 and 6, 0.25 μM Rad52; lanes 3 and 7, 0.5 μM Rad52; lanes 4 and 8, 1.0 μM Rad52. (D) Same as for (B) except 3 μM biotinated-dT30 was substituted for biotinated-dT60. Upper panel, Coomassie staining; lower panel, Western blotting. (E) The relative molar amounts of proteins bound to 3 μM biotinated-dT60 were quantified (B). Rad51 (1 μM) and/or Rad52 (1 μM) were used.