with buﬀer B and bound proteins were eluted in SDS loading buﬀer. Srs2 and PCNA were detected by Western blotting using polyclonal anti-Srs2 antibody (sc-11991, Santa Cruz Biotechnology, Heidelberg, Germany), and a polyclonal rabbit anti-PCNA serum (a generous gift from Martine Heude). RESULTS Isolation of srs2 mutations that partially suppress the UV sensitivity of rad18D mutant but do not confer UV sensitivity in otherwise wild-type cells In a search for new suppressors of the sensitivity to the alkylating agent MMS of rad18 cells, we isolated several srs2 mutants, which fell into two groups. Mutations belonging to the ﬁrst one have the same eﬀects as srs2 : they partially suppress the MMS and UV sensitivities of rad18 cells, and in the RAD18 context, the mutants are UV sensitive and show increased rates of spontaneous and UV-induced intragenic recombination. The mutations of the second group also partially suppress the sensitivities to genotoxic agents of rad18 cells but they do not confer UV sensitivity in the RAD18 context. We focused our attention on two mutants of this last class, srs2R1 and srs2R3. Figure 1A shows that they both suppress the UV sensitivity of rad18 cells, as eﬃciently as does srs2 . However, unlike srs2 cells, the single srs2R1 and srs2R3 mutants are not UV sensitive (Figure 1B). The suppression of UV sensitivity in rad18 cells is not due to a down regulation of the mutated proteins, as judged by western blot analysis (Figure 1C). As reminded in the introduction, the UV sensitivity of srs2 cells is believed to be due to the binding of recombination pro- teins to ssDNA, before and/or during replication, gener- ating toxic DNA structures if not processed by Srs2. That srs2R1 and srs2R3 are not UV sensitive suggests that these two mutated proteins are still able to remove the toxic recombination intermediates induced by UV treatment. srs2R1 and srs2R3 mutations do not show a negative interaction when combined with either rad54" or sgs1" Toxic recombination intermediates are also formed in dif- ferent double mutants involving srs2 , notably srs2 rad54 cells, which are dead, and srs2 sgs1 , which are extremely sick. We asked whether srs2R1 or srs2R3 aﬀect the viability of rad54 and sgs1 mutants, as does srs2 . We performed tetrad analysis on diploids heterozy- gous for srs2R1 and rad54 or sgs1 , and srs2R3 and rad54 or sgs1 . In these diploids, rad18 was also heterozygous but this mutation has no eﬀect on the viability of their meiotic progeny. While the negative inter- action between srs2 and rad54 or sgs1 were once more evidenced, no interaction was observed between srs2R1 or srs2R3 and rad54 or sgs1 : the double mutants rad54 srs2R1, rad54 srs2R3, sgs1 srs2R1 and sgs1 srs2R3 are viable and have no growth defects, as judged by the size of the colonies (Figure 2). Thus, Srs2R1 and Srs2R3 proteins have retained the capacity to disrupt toxic recombination structures formed not only after UV irradiation but also in the absence of Rad54 or Sgs1. Nucleic Acids Research, 2008, Vol. 36, No. 15 4967 Figure 1. UV survival of srs2R1 and srs2R3 mutants. (A) Like srs2 , srs2R1 and srs2R3 mutations partially suppress the UV sensitivity of rad18D cells. (B) srs2R1 and srs2R3 mutants are as resistant as SRS2 cells to UV, while srs2 cells are sensitive after irradiation. (C) Western blot analysis indicates that Srs2R1 and Srs2R3 are present in the same amount as Srs2 in rad18 cells. Molecular characterization of srs2R1 and srs2R3 mutations The nature of srs2R1 and srs2R3 mutations was deter- mined by DNA sequencing after PCR ampliﬁcation of the entire genes (Figure 3). srs2R3 has a G to T transversion leading to the substitution of the arginine