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

Abstract

Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G 2 /M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded(ss)DNAdependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1⌬ or rad54⌬ mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.DNA helicases perform important functions in various chromosomal transactions, including replication, repair, recombination, and transcription (1, 2). These proteins utilize the chemical energy from the hydrolysis of a nucleoside triphosphate to dissociate DNA structures and nucleoprotein complexes. Interestingly, mutations in several DNA helicases are involved in the pathogenesis of human diseases. For instance, mutations in the XPB and XPD helicases, which constitute subunits of the transcription factor TFIIH that has a dual role in nucleotide excision repair, lead to the cancer prone syndrome xeroderma pigmentosum (3). Furthermore, mutations in the BLM, WRN, and RecQ4 proteins, members of the RecQ helicase family, cause the cancer-prone Bloom, Werner, and RothmundThomson syndromes, respectively (4, 5).We are interested in the biology of various DNA helicases that influence homologous recombination and DNA repair processes. One such helicase is encoded by the Saccharomyces cerevisiae SRS2 gene, altered forms of which were first described as either suppressors of the DNA damage sensitivity of rad6 and rad18 mutants (6) or as hyperrecombination mutants (7). Detailed genetic analyses have shown that a major function of SRS2 is to attenuate homologous recombination activity to allow for the channeling of certain DNA lesions into the RAD6/ RAD18-mediated postreplication repair pathway (8, 9). Accordingly, srs2 mutants are sensitive to DNA damaging agents and show a hyperrecombination phenotype. Genetic deletion of the RAD51 or RAD52, key members of the RAD52 epis...

Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase

“…3B). Conversely, overexpression of a helicase/translocase-dead Srs2 mutant protein (Krejci et al 2004), which accumulates at the same level as wild-type protein, does not influence the level of X molecules in sgs1 mutants (data not shown). However, we note that a population of Rad51/Rad52-independent X molecules, likely representing the SCJs described in untreated wild-type cells, is also found in MMS-treated wild-type, sgs1, rad51, sgs1 rad51, and SRS2-overexpressing cells (Fig.…”

The Human F-Box DNA Helicase FBH1 Faces Saccharomyces cerevisiae Srs2 and Postreplication Repair Pathway Roles

“…An intrachromosomal recombination assay was performed using yeast strains carrying a heteroallelic duplication of LEU2, with URA3 between the LEU2 genes, as previously described (25). Spot assays were performed by evaluating cellular growth on synthetic complete medium containing adenine at a final concentration of 0.7 g/liter, with or without methyl methanesulfonate (MMS), hydroxyurea (HU), and 4-nitroquinolone-1-oxide (4-NQO; Sigma).…”

Hed1 regulates Rad51-mediated recombination via a novel mechanism

“…Based on its structural and functional similarity to BLM, Sgs1 is expected to dissociate the D-loop intermediate, disrupt the Rad51 presynaptic filament, and function with Topoisomerase III␣. Importantly, the ATPase activity of Srs2 and BLM is apparently indispensable for their recombination regulatory role (Krejci et al , 2004Wu and Hickson 2003;Bussen et al 2007).…”