4966 Nucleic Acids Research, 2008, Vol. 36, No. 15 are selected on medium lacking arginine and patched on the same medium. To determine the relative frequencies of URA3 losses, the plates are replicated on a medium con- taining 5-FOA, a drug that allows only the growth of Ura cells (23). Patches that give rise to numerous Ura papillae derive from a conversion without CO and patches with no or a very few Ura papillae reveal a CO event. This was veriﬁed by molecular analyses on a sample of convertants (21). UV treatment Stationary phase (RAD18 background) or exponentially growing (rad18 context) cells were washed in sterile water and resuspended at appropriate dilutions. UV irra- diation (254 nm) was applied after plating on YPD and survival was determined after 3 days of incubation at 308C. For spot assays, exponentially growing cells were resuspended in water at 3.106 cells/ml and 7 ml of 10-fold serial dilutions were spotted onto plates lacking leucine to select for cells that retained the plasmid. The control and UV-irradiated plates were incubated as described above. Plasmids and proteins In order to clone the mutant genes into baculoviruses, we ﬁrst constructed the p14HB-srs2R1 and p14HB-srs2R3 plasmids by the gap repair method (25). The centromeric replicative p14HB plasmid contains the whole wild-type SRS2 coding sequence and, upstream of the ORF, a BamHI site which was introduced by site-directed muta- genesis in p14H (3). A gap was made in the region of either the srs2R1 or srs2R3, and the gapped plasmids were intro- duced into the corresponding mutant cells. For srs2R1, BbvCI-gapped p14HB (13) was introduced in FF1886 and the repaired plasmid was recovered. For srs2R3, the same procedure was followed using XbaI-gapped p14HB introduced into FF1888. The BamHI-EcoRI fragment of p14HB-srs2R1 or p14HB-srs2R3 was cloned into pBacPAK-His1. The recombinant baculoviruses were constructed as recommended by the manufacturer (Invitrogen, Cergy Pontoise, France). It encodes the Srs2 proteins with 17 additional amino acid residues (MGHHHHHHVVDKLGSQM) fused to its N-terminal methionine. The wild-type fusion protein was found to complement the genotoxic sensitivity of srs2 cells (data not shown). Srs2 wild-type and mutant proteins were pro- duced and puriﬁed as described previously (13). To overproduce Srs2 wild-type and mutant proteins in yeast, the HindIII-SphI fragment of p14HB vectors was cloned into the multicopy plasmid YEp13 (26). To elim- inate the BamH1 site and restore the genuine sequence, the EagI-Bsu36I fragment that contains the BamH1 site was replaced by the corresponding fragment of p14H. The K41A mutation in the Walker A site in Srs2 was introduced by using mutagenic DNA primers and QuikChange site-directed mutagenesis kit (Stratagene, Massy, France). ATPase assay ATPase activity was measured by linking ATP hydrolysis to the oxidation of NADH as described previously (27). The dependence of the ATPase reaction on ssDNA cofac- tor (a 56 mer) was examined by the above method at 378C in a buﬀer containing 50 mM Tris–HCl pH7.6, 100 mM NaCl, 7 mM MgCl2, 5 mM DTT, 80 mgml 1 BSA and 1 mM ATP. Values for the Michaelis-Menten constants kcat and Km for ATP at saturating amount of ssDNA were derived by ﬁtting data directly to the Michaelis- Menten equation. Helicase assay A forked DNA built by the annealing of two oligonucleo- tides, P1 (50-AGAAGGTTTCGAATCAGAGGTAGGT GCCCGGCCTCCAACTTGCCGTATTCCTGGT) and Cy5-50-labeled P2 (50-Cy5-ACCAGGAATACGGCAA GTTGGAGGCCGGGCTGGATGGAGACTAAGCTT TGGAAGT) was used to assay the helicase activity of Srs2. DNA substrate (20 fmol) was incubated at 258C with 62 nM Srs2 in buﬀer A (50 mM Tris–HCl pH 7.6, 7 mM MgCl2, 5 mM DTT, 2 mM ATP and 25 mgml 1 BSA). Reactions were stopped by addition of 150 mM EDTA. The reaction products were resolved by electro- phoresis on a 8% nondenaturating polyacrylamide gel in Tris-borate-EDTA (TBE) buﬀer and quantiﬁed by ImageQuant software. Electrophoretic mobility shift assay The same forked DNA (20 fmol) used in helicase assay was incubated with increasing amount of Srs2 protein in buﬀer A without ATP for 30 min at 258C. Gel shift was detected after electrophoresis on a 6% nondenaturat- ing polyacrylamide gel using a Storm 960 apparatus (GE Healthcare Biosciences, Saclay, France). Electron microscopy (EM) Rad51 ﬁlaments on ssDNA were formed by incubation at 378C of 3.5 mM Rad51 protein in 42 mM MOPS pH 7.2, 3 mM Mg(OAc)2, 1 mM DTT, 20 mM NaCl, 2.5 mM ATP with 11 mM (ntd) X174 viral (+) strand for 3 min fol- lowed by addition of 330 nM RPA and subsequent incu- bation for 15 min. Five microliters of this reaction was mixed with 5 ml of various amount of Srs2 diluted in 20 mM phosphate buﬀer pH 7.8, 200 mM NaCl, 10% gly- cerol and 1 mM ß-mercaptoethanol and incubated for 10 min at 378C. The reaction mixtures were then diluted 20-fold in 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2 without any chemical ﬁxation and analyzed by EM as previously described (28). Immunoprecipitation For immunoprecipitation assays, yeast native extracts from FF182029 treated with 0.3% MMS were prepared as described previously (18). Extract (2.5 mg of protein) was incubated overnight in buﬀer B (50 mM Tris–HCl pH 7.4, 150 mM NaCl and supplemented with protease inhib- itors) at 48C with 1.7 mg of puriﬁed HisSrs2, HisSrs2R1 or HisSrs2R3 protein and 1 mg of antibody against His- tag (Roche Diagnostics, Meylan, France). Prewashed protein G agarose beads (Roche Diagnostics) were then introduced for 5 hrs at 48C. Beads were washed six times