ATPase and DNA Helicase Activities of the Saccharomyces cerevisiae Anti-recombinase Srs2

Abstract

Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination. Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants. Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2. ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard. Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation. In its helicase action, Srs2 prefers substrates with a 3 ss overhang, and ϳ10 bases of 3 overhanging DNA is needed for efficient targeting of Srs2 to the substrate. Even though a 3 overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein. The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the singlestrand DNA-binding factor replication protein A.DNA helicases are ubiquitous among prokaryotes and eukaryotes. These enzymes utilize the free energy derived from the hydrolysis of a nucleoside triphosphate to disrupt the hydrogen bonds that bind the complementary strands of duplex DNA. DNA helicases play an essential role in virtually every aspect of nucleic acid metabolism, including transcription, replication, recombination, and repair (reviewed in 1-3).We focus on the mechanism of homologous recombination in eukaryotes and are interested in the biology of DNA helicases that influence recombination events. One such helicase is encoded by the Saccharomyces cerevisiae SRS2 1 (Suppressor of RAD Six-screen mutant 2) gene. Srs2 protein belongs to the SF1 helicase family and contains regions of similarity to the bacterial UvrD, Rep, and PcrA helicases (4). A mutant form of SRS2 was initially identified as a suppressor of the radiationsensitivity of rad6 and rad18 mutants that are defective in post-replicative DNA repair (4, 5). Subsequent studies revealed that suppression of rad6 and rad18 mutations by the srs2 mutation is due to heightened recombination mediated by the RAD52 epistasis group of genes (6). Consistent with this observation, srs2 mutants show a hyper-recombination phenotype (7,8). Together, these genetic observations indicate that SRS2 negatively regulates RAD52-mediated homologous recombination. It has been suggested that by antagonizing the recombination machinery, Srs2 ensures the channelling of DNA lesions that arise during DNA replication into the Rad6/Rad18-mediated repair reactions (4). Hence, in rad6 and rad18 mutants, repair of the replication-associated DNA lesions by recombination is inefficient unless SRS2 is inactivat...

Role of ATP Hydrolysis in the Antirecombinase Function of Saccharomyces cerevisiae Srs2 Protein

“…This expectation was confirmed by examining the ATPase activity of purified proteins with [␣-32 P] ATP and thin layer chromatography (14,23). We showed previously that ATP hydrolysis by Srs2 occurs only in the presence of DNA with ssDNA being much more effective than dsDNA in this regard (9,23).…”

“…As summarized in Fig. 2A, although robust ATPase activity was observed with wild-type Srs2 in the presence of ssDNA (14,23), the two srs2 mutant proteins showed less than 1% of the wild-type level of ATP hydrolysis. Likewise, no significant ATP hydrolysis by either of the srs2 mutants was seen when the ssDNA was omitted or substituted with dsDNA (data not shown).…”

“…DNA Substrates-The H2 and D1 oligonucleotides used in the construction of the helicase substrate have been described (23). Oligo-1 used in the DNA binding experiments has also been described (25).…”

“…Srs2 Expression and Purification-srs2 K41A and srs2 K41R proteins were overexpressed in Escherichia coli cells and purified to near homogeneity as described previously for wild-type Srs2 (14,23). Rad51 was overexpressed in yeast and purified to near homogeneity as described (16).…”

“…The DNA helicase substrate was obtained by heating equimolar amounts of H2 and radiolabeled D1 oligonucleotides to 95°C for 10 min in buffer B (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 100 mM NaCl), followed by slow cooling to room temperature. The annealed substrate was purified from a 12% non-denaturing polyacrylamide gel, as described (23). The X174 (ϩ) strand was purchased from New England Biolabs.…”