Figure 4.—

Rad52-L89F-Rad59 interaction. (A) SDS-PAGE analysis of Rad59 protein in wild-type, rad52Δ, and rad52-L89F strains. Total protein extract (5 μg) was loaded for each strain. Coomassie staining (top) and Western blot using αRad59 polyclonal antibody (bottom) are shown. The position in the gel at which Rad59 migrates is indicated. (B) Purification of Rad59-GST fusion protein in wild-type, rad52Δ, and rad52-L89F strains. Purification of GST in the wild type was included as a negative control. Protein expression was under the control of the CUP1 promoter and was induced by addition of CuSO₄ to a final concentration of 0.5 mm. Coomassie staining (top) and Western blot using αRad52 polyclonal antibody (bottom) of total cell extracts (left) and purified fractions (right) are shown. The positions in the gel at which Rad59-GST, Rad52, and GST migrate are indicated. For the preparation of extracts, cells were grown in 500 ml of SC-Leu to an OD_660nm of 0.8. Cells were harvested, washed with water, and resuspended in one pellet volume of 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 4 mm MgCl₂, 5 mm DTT, 10% glycerol, 1 m NaCl. Leupeptin and pepstatin A were added to a final concentration of 2 μg/ml and 1 μg/ml, respectively. Extracts were made with glass beads (McCraith and Phizicky 1990), followed by supplementation with 1 mm PMSF and 30 min centrifugation at 14,000 rpm in a JA-20 Beckman rotor. For Rad59::GST protein purification, supernatant was incubated with 1/100 volume of glutathione-Sepharose 4B (A. P. Biotech) previously equilibrated in 50 mm potassium phosphate pH 7.2, 1 mm DTT, 0.5 mm EDTA, 10% glycerol, 1% Triton X-100, 1 m NaCl. Samples were washed twice with 500 bead volume of 50 mm potassium phosphate pH 7.2, 1 mm DTT, 0.5 mm EDTA, 10% glycerol, 1% Triton X-100, 0.5 m NaCl. Proteins were eluted by boiling in 1× loading buffer for 5 min.