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J. Biol. Chem., Vol. 279, Issue 22, 23193-23199, May 28, 2004

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

Lumir Krejci, Margaret Macris, Ying Li, Stephen Van Komen, Jana Villemain¶, Thomas Ellenberger, Hannah Klein||, and Patrick Sung**

From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, the ^¶Institute of Biotechnology and Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, and the ^||Department of Biochemistry, New York University School of Medicine, New York, New York 10016

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 G₂/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)DNA-dependent 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.

Received for publication, March 8, 2004

^* This work was supported by National Institutes of Health Grants ES07061, GM57814, and GM53738, by Department of Energy Grant DE-FG02-01ER63071, by National Institutes of Health Postdoctoral Fellowship F32GM065746, and by Department of Defense Postdoctoral Fellowship BC020457. The molecular electron microscopy facility at Harvard Medical School was established by a donation from the Giovanni Armeise Harvard Center for Structural Biology and is maintained through a National Institutes of Health grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar St., SHM C130A, New Haven, CT 06520. Tel.: 203-785-4553; Fax: 203-785-6037; E-mail: Patrick.Sung{at}yale.edu.

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