RAD51, RAD52, RAD55 or RAD57 genes [(7) and our unpublished data] that are all involved in the formation/ stabilization of presynaptic Rad51 nucleoﬁlaments. This epistasis of rad51 over srs2, together with data showing that Srs2 prevents recombinational repair in cells contain- ing leaky alleles of RAD51 or RAD52 (7–10), led to the proposal that Srs2 eliminates toxic recombination inter- mediates (11) rather than preventing their formation. This hypothesis was later supported by the ability of Srs2 to dismantle Rad51 nucleoprotein ﬁlaments formed in vitro on single-stranded DNA (ssDNA) through its translocase activity (12,13). In the absence of any treat- ment, toxic recombination intermediates are also formed in double mutants involving srs2 and mutations aﬀect- ing recombination and/or replication such as rad54 and sgs1 . The synthetic lethality or sickness of the double mutants is suppressed by a third mutation in RAD51, RAD52, RAD55 or RAD57 (10,14,15)). Recently, it was proposed that not only the translocase but also the heli- case activity of Srs2 may reverse nonproductive recombi- nation intermediates resulting from strand invasion of the homologous duplex DNA (16). While the deletion of SRS2 sensitizes wild-type cells to UV, it increases the resistance of the highly UV sensitive rad6 and rad18 mutants. In these PRR-deﬁcient contexts, it has been demonstrated that the high sensitivities of rad6 and rad18 cells is related to the prevention of recombina- tional repair by Srs2 which is recruited by a sumoylated form of PCNA (17,18). Indeed, deletion of the Small Ubiquitin-related Modiﬁer (SUMO)-speciﬁc ligase gene SIZ1, which is responsible for the sumoylation of PCNA on the K164 lysine or PCNA mutations that prevent its sumoylation suppress DNA damage sensitivities of rad18 or rad6 cells, as does the deletion of SRS2 (17–19). Srs2 was also shown to be involved in crossover (CO) control and proposed to unwind the invading strand of recombination intermediates, allowing conversion but pre- venting formation of COs (20,21). This role of Srs2 would involve the helicase activity, and appears to be dependent on PCNA sumoylation (21). These genetic data show that Srs2 plays diﬀerent roles. One is to eliminate toxic recombination intermediates. Importantly, in wild-type cells, a SIZ1 deletion has no signiﬁcant eﬀect on repair, mutagenesis or recombination (18,19), suggesting that elimination of toxic intermediates does not depend on PCNA sumoylation. Another role of Srs2 is to prevent recombinational repair. This has been evidenced only in PRR-deﬁcient contexts, and the extra- polation of this activity to wild-type cells remains ques- tionable in view of the absence of eﬀects of the single siz1 mutation. In a hope to obtain mutations that separate the func- tions, we screened for new Srs2 mutations suppressing the MMS sensitivity of rad18 cells. We describe here two of them which confer UV resistance to rad18 cells but are not by themselves UV or MMS sensitive. However, they do have an eﬀect on the incidence of reciprocal recombina- tion associated to gene conversion events. The biochem- ical study of the puriﬁed mutant proteins indicates that the mutations aﬀect diﬀerently the activities of Srs2, although their biological eﬀects are very similar. Nucleic Acids Research, 2008, Vol. 36, No. 15 4965 MATERIALS AND METHODS Yeast strains and genetic analysis Except for CO experiments, the strain used in this study are isogenic derivatives of either FF1852 (MATa leu2-3,112 trp1-289 ura3-52 ade5) or of FF18733 (MATa leu2-3,112 trp1-289 ura3-52 his7-2 lys1-1). The MATa haploids in the FF1852 series are: FF1886, rad18::LEU2 srs2R1; FF1888, rad18::LEU2 srs2R3; FF18672, srs2::LEU2; D84-3B, siz1::KAN; D48-3C, srs2R1; D52-2C, srs2R3; the MATa in the same background is FF18238, rad18::LEU2. The MATa haploids in the FF18733 series are FF182029, pol30-K127R; FF182031, pol30-K127/164R; FF182027, pol30-K164R; D83-5B, rad18::KAN srs2::KAN. In the same background, the MATa strains are FF181496, sgs1::URA3; FF18974, rad54::LEU2. For CO experiments, the strains used are derivatives of W303, as were the strains used in our previous experi- ments on COs (21). The wild-type SRS2 and the mutants srs2R1 and srs2R3 are tagged by HA. We found no eﬀect of the HA tag on the resistance to genotoxic agents, indi- cating that the Srs2 proteins are functional. The ‘CY2715’ wild-type HA-SRS2 strain (22) was a gift of Dr Foiani. HA-SRS2 was replaced by HA-srs2R1 or srs2R3 by the pop-in/pop-out method. For this, srs2R1 and srs2R3 were cloned into the URA3 pRS406 vector. The linearized plas- mids were integrated into the HA-SRS2 strain, and pop- out events were selected on 5-ﬂuoroorotic acid (5FOA), a drug that allows only the growth of Ura cells (23). The presence of the srs2R1 or srs2R3 mutation was tested by the suppression of rad18 MMS sensitivity in meiotic analysis of diploids obtained by crossing the strains with rad18 haploids. It was conﬁrmed by DNA sequencing. The strains were crossed with the srs2D haploid bearing the ectopic recombination system (D498-1C) to isolate from the meiotic progeny the wild-type strain ‘D508- 10C’ of genotype MATa arg 4-Bg URA3::arg4- RV::ura3- 1 ade2-1 trp1-1 leu2-3,112 his3-11,15 can1-100 HA3SRS2::srs2. The corresponding HA-srs2R1 and HA- srs2R3 strains are ‘D511-4D’ and ‘D509-8B’, respectively. Genetic analyses were performed according to published procedures (24). Determination of CO frequencies This analysis was performed as previously described (21). Brieﬂy, CO frequencies associated to gene conversion events were determined in haploid cells carrying two arg4 alleles mutated at diﬀerent sites and located on dif- ferent chromosomes. One allele is at its endogenous loca- tion on chromosome VIII, and the other one is located on chromosome V, between a wild-type and a mutated allele of URA3 in direct orientations. When gene conversion occurs without CO, the two URA3 and ura3-1 genes remain in the parental direct repeat conﬁguration in which loss of URA3 is a frequent event. A CO associated to a conversion of an arg4 allele generates a reciprocal translocation and a separation of the URA3 and ura3-1 alleles on each of the translocated chromosomes. In this situation, loss of the URA3 information is much less fre- quent. Spontaneous and independent Arg+ convertants