4972 Nucleic Acids Research, 2008, Vol. 36, No. 15 suppresses the sensitivity of a subpopulation of cells (5), while in PRR-proﬁcient cells, it sensitizes a subpopulation of cells (3). It is now understood that these eﬀects reﬂect two roles of Srs2. One is the elimination of potentially toxic structures formed by recombination proteins (11), and a second role is the prevention of recombinational repair through its recruitment by sumoylated PCNA, evi- denced only in PRR-deﬁcient cells (17,18). The ability of Srs2 to disrupt in vitro Rad51 nucleoﬁlaments on ssDNA would be suﬃcient to account for the elimination of both the toxic and the recombination intermediates. It is likely that toxic structures are also formed after UV treatment of rad18 srs2 cells, but this cannot be directly tested because rad51, as well as other mutants aﬀected in the formation of Rad51 nucleoﬁlaments, has a synergistic eﬀect with rad18 on UV survival, regardless of the status of Srs2. In this study we describe two new srs2 mutants that have acquired the ability to perform recombinational repair in a rad18 context, but have retained the ability to reverse toxic structures in a RAD18 context. The biochem- ical activities of the two mutant proteins are diﬀerently aﬀected. The only known defect of Srs2R1 is the loss of interaction with sumoylated PCNA. This explains its sup- pressive eﬀect on the UV sensitivity of rad18 cells. The mutation is located in the C-terminal domain of the pro- tein, known to interact with sumoylated PCNA, and dele- tions in this domain were reported to suppress the sensitivity of PRR mutants (18). Diﬀerently, Srs2R3 inter- acts as eﬃciently as the wild-type protein with sumoylated PCNA and its cellular amount in the cells is not decreased. However, the ATPase and helicase activities of the pro- tein, as well as its ability to disrupt Rad51 nucleoprotein ﬁlaments, are decreased. We suggest that the inability of Srs2R3 to prevent recombinational repair in rad18 cells may be related to the size of the Rad51 presynaptic ﬁla- ment that must be removed. Indeed, EM analyses revealed that UV lesions, if not repaired, induce uncoupling of nascent strands synthesis at replication forks leading to up to 3 kb long ssDNA regions (33). In rad18 cells, only recombination could save the cells, but Srs2 counteracts this repair. Because of its weak translocase activity, Srs2R3 would be unable to fully destroy the Rad51 nucleoﬁlaments, allowing recombinational repair to occur. This interpretation ﬁts with our data showing that overexpression of Srs2R3 in rad18 srs2 cells largely sensitizes the cells. It ﬁts also with the semi dom- inance of srs2 mutants in rad18 homozygous diploids: the cells are more sensitive with two copies of SRS2 than with a single one, indicating a gene dosage eﬀect (34). Removal of toxic recombination structures by Srs2 does not require an interaction with sumoylated PCNA Contrarily to srs2 cells, the two mutants srs2R1 and srs2R3 are not UV sensitive. This indicates that the two mutated proteins have retained the ability to reverse toxic recombination structures. The Srs2R3 protein is therefore in suﬃcient amount to perform this activity. It suggests that in PRR proﬁcient cells, these structures are limited in size and/or number. The only deﬁcient activity that we observed for Srs2R1 is its binding to sumoylated PCNA. This interaction is therefore not required to remove the dead-end structures. In agreement with this conclusion, the single siz1 mutant, deﬁcient in sumoylation of the lysine 164 of PCNA, shows no UV sensitivity (18). We asked if the potentially toxic intermediates formed in the rad54 or sgs1 mutants, and revealed by their Rad51- dependent synthetic lethality with srs2 , depends on PCNA sumoylation. The srs2R1 mutation has no eﬀect on the growth of rad54 or sgs1 cells. Thus, the toxic struc- tures formed in these mutants, as those formed after UV treatment, are reversed by Srs2 independently of an inter- action with sumoylated PCNA. In these cases, the recruit- ment of Srs2 could be mediated through its interaction with Rad51 (12). Control of COs involves the helicase activity of Srs2 and is partially dependent of its interaction with sumoylated PCNA It has been proposed that Srs2 prevents COs by promoting SDSA, a recombination pathway non-associated with COs (20,21). This would imply the unwinding activity of Srs2 in order to displace the elongated invading strand from the D-loop. We recently showed that Srs2 has the biochemical activities that are required to perform SDSA (16). However, in vivo evidence for an implication of the helicase function of Srs2 in this process is missing. We hypothesized that the srs2R3 mutation which decreases the helicase activity of Srs2 would increase the percentage of CO associated to conversion events. We indeed found that in this mutant CO frequency is increased from 14% in wild-type cells to 33%, while it reaches 50% in the absence of Srs2. The approximate correlation between the level of COs and that of the helicase activity supports the idea that Srs2 controls the level of COs through its helicase activity. However, we cannot exclude the possibility that the increased CO frequency observed in srs2R3 cells relates to its decreased DNA binding ability and translocase activity. An srs2 mutant aﬀected speciﬁcally in its unwind- ing activity would be required to answer this question. The Srs2R1 mutation increases the frequency of COs associated with conversions from 14% in wild-type cells to 25%. This indicates that the interaction with sumoy- lated PCNA is involved in CO control. However, since the percentage of COs in srs2R1 mutant is half that in srs2D cells, it suggests that the enrollment of the Srs2 helicase to disrupt D-loops is only partially dependent on sumoy- lated PCNA. It is important to note that the eﬀect on CO incidence of srs2R1 and of srs2R3 is the only phenotype of these mutants in a RAD18 context. In both cases, it is likely due to a decreased eﬃciency in the processing of recombi- nation intermediates into the SDSA pathway. This would be related to a limited Srs2 helicase activity, for diﬀerent reasons in each of the mutants. What is the role of PCNA sumoylation? The absence of genetic eﬀects of the single siz1 or srs2R1 mutation raises the question of the role of PCNA sumoy- lation at K164 in wild-type cells. Our favorite model is the