Nucleic Acids Research, 2008, Vol. 36, No. 15 4971 Figure 7. Overexpression of Srs2R1 and Srs2R3 sensitizes rad18 srs2 cells (A) but not pol30-K127/164R cells (B) to UV radiation. Ten-fold serial dilutions of an equal number of exponentially phase growing cells were spotted onto plates and irradiated with ultraviolet light at indicated doses. The proposed interpretation is that Srs2 acts on mitotic recombination intermediates at a postsynaptic stage. It unwinds the invading and elongated strand from its tem- plate, promoting synthesis-dependent strand annealing (SDSA), a mechanism that allows gene conversion but not associated COs. A partially unanswered question con- cerns the recruitment of Srs2 to the D-loop. Using a genetic system allowing to determine the frequency of COs associated to a selectable conversion event involving ectopic heteroalleles (see Materials and methods section), we previously showed (21) that CO frequencies rise from 11% in wild-type cells to 50% in both the srs2 mutant and in the pol30-K127/164R mutant that can neither be sumoylated nor ubiquitinated at the lysine 164 of PCNA (31). This result strongly suggested that SUMO-modiﬁed PCNA recruits Srs2 into the D-loop. However, because the double PCNA mutant is also deﬁcient in PRR, the high incidence of COs observed in this strain is possibly also due to the PRR deﬁciency, and not only to the lack of Srs2 recruitment by PCNA. To address this question, we used the srs2R1 mutation, which prevents the interac- tion of the helicase with PCNA. We introduced at their native locus a HA-tagged version of SRS2 or of srs2R1 into the strain used by Robert et al. (21). The percentage of COs associated with spontaneous gene conversion events was found to be increased 1.8-fold in srs2R1 as compared with SRS2 cells (25 and 14%, respectively) (Figure 8). This result shows that the interaction between SUMO-modiﬁed PCNA and Srs2 is indeed involved in CO control. However, the level of COs measured in srs2R1 (25%) is half that in srs2 or in the double PCNA mutants (50%). It suggests that Srs2 controls CO both in a PCNA-dependent and independent fashion. We cannot formally exclude that a possible residual inter- action between PCNA and Srs2R1 could account for Figure 8. CO bias observed in srs2 mutants. The percentage of CO was determined using three individual segregants for each genotype. this result. The same experiment was carried out with srs2R3. Interestingly, this mutant leads to a 2.3-fold increase in COs over the wild-type level (33 and 14%, respectively). The percentage is still lower than that observed in srs2 mutants. The eﬀect of this mutation likely results from the decreased helicase activity of Srs2R3, and thus from less eﬃcient repair through the SDSA pathway. DISCUSSION Early studies on srs2 mutations revealed two opposite eﬀects with respect to radiations. In the highly sensitive rad6 or rad18 haploid mutants, deﬁcient for PRR, srs2