Rad52 SUMOylation affects its biochemical activities. ( A ) SUMOylation of Rad52 inhibits its binding to DNA. Increasing amounts of Rad52 or Smt3-Rad52 (20, 40, 80, 200 nM) were incubated with fluorescently labelled 49-mer ssDNA (0.49 µM nucleotides) at 37°C for 10 min. The reaction mixtures were resolved in 7.5% native polyacrylamide gels, and the DNA species were quantified using Quantity One software (Bio-Rad). ( B ) The results from (A) plotted. ( C ) SUMOylation of Rad52 inhibits its strand annealing activity. Labelled Oligo-1 (0.25 µM nucleotides) and Oligo-2 (0.25 µM nucleotides) were incubated separately with RPA (20 nM) for 3 min at 37°C. The annealing reactions were initiated by mixing RPA-coated oligonucleotides and Rad52 or Smt3-Rad52 proteins (0.7, 5, 10, 20 nM) and incubated at 37°C. After 8 min of incubation, 9 µl of the annealing reactions was removed and treated with 0.5% SDS, and 500 µg/ml proteinase K at 37°C for 10 min. The reaction mixtures were resolved in 12% native polyacrylamide gels. ( D ) The averaged values of results from three independent experiments are plotted.