FIGURE 5.

Rad52 stimulates endonuclease activities of Dna2 and Rad27. A, the pRS325-ADH1 plasmids expressing Dna2, Rad52, and Rad52-QDDD/AAAA were introduced into YJA1BJK65. None, empty vector (negative control). Transformants were grown and spotted as described in the legend for Fig. 1A. B, expression levels of wild type (Rad52) and mutant (Rad52-QDDD/AAAA) proteins were examined as described in the legend for Fig. 1B. C, SDS-PAGE analysis of purified recombinant Rad52 and Rad52-QDDD/AAAA proteins. Both proteins were purified as described under “Materials and Methods.” D and E, purified Rad52 proteins stimulate endonuclease activities of Dna2 and Rad27 in vitro. The substrates used in this experiment are indicated at the right of each panel. The asterisk represents the position of ³²P radiolabel. The reaction mixtures were assembled with components as indicated above the gel and incubated at 37 °C for 30 min. The additions and omissions are indicated + and − signs, respectively. Cleavage products were analyzed on a 10% denaturing polyacrylamide gel and quantified as described under “Materials and Methods.” The stimulation fold obtained (with respect to Dna2 or Rad27 alone) by the addition of Rad52 proteins is indicated below the gel.