4970 Nucleic Acids Research, 2008, Vol. 36, No. 15 Figure 5. Srs2R3 disrupts the Rad51 presynaptic ﬁlament less eﬃciently than Srs2 and Srs2R1. (A) Rad51-ssDNA nucleoprotein ﬁlament. (B) Preformed Rad51-ssDNA complexes were incubated for 10 min with Srs2. The arrows point to the ssDNA covered with RPA. (C) Blow- up of ssDNA covered with RPA. (D) The percentage of disrupted Rad51 presynaptic ﬁlament was determined for various amounts of helicases. For each concentration, 300–500 molecules were counted. Scale bars, 50 nm. two independent studies (17,18) showing an important interaction of Srs2 with non-sumoylated PCNA. The reason for this discrepancy is not clear, it could reﬂect small diﬀerences in experimental procedures. However, as we will show below, the interaction between Srs2 and unmodiﬁed PCNA does not seem to play a role in vivo. Overexpression of Srs2R1 and Srs2R3 sensitizes rad18D srs2D but not pol30-K127/164R cells to UV radiation Although the Srs2R3 protein still interacts with K164- sumoylated PCNA, the mutation suppresses the UV sen- sitivity of rad18 cells to the same extent as does the deletion of SRS2 (Figure 1A). Since the diﬀerent activities of Srs2 tested are present but less eﬃcient in srs2R3, we predicted that overexpressing the mutant protein would sensitize the double mutant rad18 srs2 cells. To test this idea, we introduced SRS2 or srs2R3 under their own promoter into the multicopy yeast vector YEp13. The rad18 srs2 double mutant cells were transformed and UV survival assays performed. As predicted, overex- pression of srs2R3 strongly reduces UV survival of these cells, and furthermore, to a similar extent as does the wild- type SRS2 overexpression (Figure 7A). In the same con- ditions, overexpression of the Srs2K41A helicase- and ATPase-dead mutant protein (32) has no eﬀect Figure 6. Srs2R1 does not interact with sumoylated PCNA. Immunoprecipitation experiments were performed with total cell lysates prepared from pol30-K127R mutant treated with 0.3% MMS to gen- erate maximal levels of SUMO-modiﬁed PCNA. Input and bound frac- tions were analyzed by western blot as indicated. (Figure 7A), indicating that indeed the srs2R3 overexpres- sion eﬀect relates to an increased activity and not simply to the abnormally high protein concentration. These results support the view that a single copy of srs2R3 does not produce enough protein to prevent recombina- tional repair in UV-treated rad18 cells, and consequently that the Srs2R3 protein suﬀers from a decreased speciﬁc activity rather than from a complete deﬁciency in a particular activity. When we did the same experiment with srs2R1, we also observed that its overexpression sensitizes rad18 srs2 cells (Figure 7A). One possible explanation for this result is that a residual interaction (although not detected in our experiments) between the overproduced Srs2R1 proteins and K164-sumoylated PCNA allows enough helicase recruitment to prevent recombinational repair. A second explanation is that overproduction of Srs2 prevents recombinational repair by a recruitment through unmodi- ﬁed PCNA or directly through its interaction with Rad51 (12). In order to diﬀerentiate between these possibilities, srs2R1 was overexpressed in the pol30-K127/164R mutant unable to be sumoylated on lysine 127 or sumoylated or ubiquitinated on lysine 164. Overproduction of Srs2R1 does not sensitize the pol30-K127/164R mutant to UV (Figure 7B). Similar results are obtained when wild-type SRS2 or srs2R3 were overexpressed. Altogether, these data indicate that, at least in a context where PCNA cannot be ubiquitinated, the recruitment of Srs2 to pre- vent recombinational repair is strictly dependent upon its interaction with sumoylated PCNA. Srs2R1 and Srs2R3 mutants show increased CO frequencies The absence of Srs2 was shown to enhance both DSB-induced and spontaneous CO frequencies (20,21).