4968 Nucleic Acids Research, 2008, Vol. 36, No. 15 Figure 2. srs2R1 and srs2R3 do not aﬀect the viability of rad54 or sgs1 cells. Two tetrads from crosses indicated above each picture are shown. Circles and squares indicate the rad54 srs2 and sgs1 srs2 combinations, respectively. (A) While rad54 srs2 cells are dead, rad54 srs2R1 and rad54 srs2R3 double mutants are perfectly viable. (B) The severe growth defect seen in sgs1 srs2 cells is not observed in either sgs1 srs2R1 or sgs1 srs2R3 double mutants. Figure 3. (A) Localization of srs2R1 and srs2R3 mutations. (B) Modiﬁ- cations induced by mutations srs2R1 and srs2R3 at the protein level. in position 337 by a serine. This mutation is close to the helicase domain IV (amino acid 310 to 321). srs2R1 muta- tion is the addition of one adenine in position 3480. The resulting protein is shorter by six amino acids and ﬁve of the last six amino acids of the C-terminal end are changed. This modiﬁcation occurs in the interacting domain with sumoylated PCNA (18). Unexpectedly, although the srs2R1 and srs2R3 muta- tions were located in a diﬀerent region of the gene, the mutants have similar phenotypes. In order to determine the defective properties of these mutants, we puriﬁed the mutated proteins and compared their biochemical proper- ties to those of the wild-type protein. Unlike Srs2R1, Srs2R3 is affected for all of its biochemical functions Srs2 is a ssDNA-dependent ATPase (12,13,29). The ATP hydrolysis is believed to reﬂect its translocase activity Table 1. Km and kcat values of the enzymes for ATP Protein Srs2 Srs2R1 Srs2R3 Km (mM) 143 94 19 kcat (min 1) 3316 2278 373 on ssDNA. Therefore, we determined the kcat and the Km for ATP of the wild-type and the two mutated Srs2 proteins, using a 56-mer oligonucleotide as ssDNA cofactor (Table 1). The ATPase activity was determined with saturating amounts of ssDNA (data not shown) and, therefore, represents the true catalytic activity once the protein is bound to the ssDNA. Srs2R1 binds and hydro- lyzes ATP as eﬃciently as Srs2 (Km 94 mM versus 143 mM and kcat 2778 min 1 versus 3316 min 1). These kcat values are in the same range that the one previously reported measured with X viral (+) strand as cofactor (29). Unlike Srs2R1, Srs2R3 is strongly aﬀected for ATP hydrolysis, with only about 10% of the wild-type activity (kcat 373 min 1 versus 3316 min 1). Surprisingly, Srs2R3 binds more eﬃciently ATP than the wild-type protein (Km 19 mM versus 143 mM). However, the ATP intracellular concentration in yeast is supposed to be superior than 1 mM (30), a much higher value than the calculated Km of Srs2 for ATP. It indicates that the ATP binding para- meter can be disregarded for the in vivo function. We next studied the DNA helicase activities of the two Srs2 mutant proteins, using a 50-Cy5-labeled forked DNA containing a 30-bp duplex region ﬂanked by two single- stranded tails of 25 nucleotides. As shown in Figure 4A and B, the wild-type Srs2 and the mutated Srs2R1 proteins have a similar helicase activity, while Srs2R3 unwinds poorly the DNA substrate. The attenuated helicase ability of Srs2R3 may result from an ATPase defect and/or from a default in DNA binding. For this reason, we compared the DNA binding abilities of Srs2R3 and of the wild-type protein with a DNA mobility shift assay. We incubated increasing amounts of wild-type Srs2 and Srs2R3 with the same DNA substrate used in the helicase assay fol- lowed by resolution of the reaction mixtures in nondena- turating polyacrylamide gels. As shown in Figure 4C, Srs2R3 binds less eﬃciently forked DNA than wild-type Srs2. At 140 nM protein, almost all DNA was bound to Srs2 while <50% of it was bound by Srs2R3. Srs2R3, but not Srs2R1, disrupts less efficiently the Rad51 presynaptic filament Srs2 is able to disrupt Rad51 presynaptic ﬁlaments (12,13). Because srs2R1 and srs2R3 mutants are not UV sensitive (Figure 1), we hypothesized that Srs2R1 and Srs2R3 remove toxic recombination intermediates by releasing Rad51 from the nucleoprotein ﬁlament. EM was used to monitor this activity for both mutated and wild-type proteins. Rad51 was ﬁrst incubated for 3 min with X174 ssDNA followed by addition of RPA and subsequent incubation for another 15 min in order to assemble Rad51 nucleoprotein ﬁlaments. As a function