Nucleic Acids Research, 2008, Vol. 36, No. 15 4969 Figure 4. Srs2R3 is defective in both helicase activity and DNA binding. (A) Kinetics of helicase activity of wild-type and mutated Srs2. (B) Quantiﬁcation of experiments showed in (A). Each point represents the average of three independent assays. Error bars show the standard deviation. (C) Srs2R3 binds forked DNA less eﬃciently than Srs2 as observed by gel shift assay. of the amount of Srs2 introduced into the reaction, a progressive loss of Rad51 ﬁlaments, coupled with a con- comitant formation of RPA-ssDNA complexes, was observed (Figure 5). At 120 nM Srs2 or Srs2R1, almost all the observed DNA molecules were covered by RPA while, at the same concentration, <5% of the Rad51 nucleoprotein ﬁlaments were destabilized by Srs2R3. 400 nM of Srs2 were required to obtain disruption of all Rad51 ﬁlaments. Because disassembly of the presynaptic ﬁlament requires the translocase activity of Srs2 on ssDNA, a function that requires ATP hydrolysis, the weak capacity of Srs2R3 protein to remove Rad51 from ssDNA may result from two failing activities: a reduced ability to bind ssDNA and/or a weak ATPase activity. Taken together, the results from biochemical and EM analyses clearly indicate an attenuated activity for all the tested functions of Srs2R3, while Srs2R1 behaves like the wild-type Srs2 protein. Srs2R1 does not bind SUMO-modified PCNA It was previously reported that Srs2 preferentially inter- acts with the sumoylated forms of PCNA (17,18). This interaction involves the C-terminal 138 residues of Srs2 protein, but a C-terminal truncation of the last six amino acids strongly reduces the two-hybrid interaction with both SUMO and PCNA. The Srs2R1 is modiﬁed in its C-terminal domain. The protein lost its six last residues and 5 of the six last amino acids are changed (Figure 3). Therefore, we asked if the ability of Srs2R1 to interact with sumoylated PCNA is aﬀected. Puriﬁed His-tagged Srs2 wild-type or mutated proteins were incubated with total cell extracts in order to examine their binding to PCNA and sumoylated PCNA. Extracts were prepared from a pol30-K127R mutant treated with 0.3% MMS before lysis to induce extensive SUMO conjugation at lysine 164 (K164) (31). The pol30-K127R mutant was used because this lysine was shown to be sumoylated to a small extent in a Siz1- independent manner, a modiﬁcation that plays a minor biological role (17,18). Under these conditions, the HisSrs2 and HisSrs2R3 recombinant proteins clearly bound the K164-sumoylated PCNA while no interaction could be detected with the HisSrs2R1 recombinant protein (Figure 6). Interestingly, we detected a very faint interac- tion between unmodiﬁed PCNA and Srs2 and no interac- tion between unmodiﬁed PCNA and either Srs2R1 or Srs2R3, even with longer exposure of the blot (data not shown). These data diﬀer from results found previously in