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Genomic Screening in Vivo Reveals the Role Played by Vacuolar H⁺ ATPase and Cytosolic Acidification in Sensitivity to DNA-Damaging Agents Such as Cisplatin

Pharmaceutical Science Research Division (C.L., B.H., B.P.) and Genomics Facility (M.J.A.), School of Biomedical and Health Sciences, King's College London, London, United Kingdom

Screening the Saccharomyces cerevisiae homozygous diploid deletion library against a sublethal concentration of cisplatin revealed 76 strains sensitive to the drug. As expected, the largest category of deletions, representing 40% of the sensitive strains, was composed of strains lacking genes involved in DNA replication and damage repair. Deletions lacking function of the highly conserved vacuolar H⁺ translocating ATPase (V-ATPase) composed the category representing the second largest number of sensitive strains. The effect on cell death exhibited by V-ATPase mutants was found to be a general response to various DNA damaging agents as opposed to being specific to cisplatin, as evidenced by sensitivity of the mutants to hydroxyurea (a DNA-alkylating agent) and UV irradiation. Loss of V-ATPase does not affect DNA repair, because double mutants defective for V-ATPase function and DNA repair pathways were more sensitive to cisplatin than the single mutants. V-ATPase mutants are more prone to DNA damage than wild-type cells, indicated by enhanced activation of the DNA damage checkpoint. Vacuole function per se is not cisplatin-sensitive, because vacuolar morphology and vacuolar acidification were unaffected by cisplatin in wild-type cells. V-ATPase also controls cytoplasmic pH, so the enhanced sensitivity to DNA damage may be associated with the drop in pHi associated with V-ATPase mutants. The increased loss in cell viability induced by cisplatin at lower pH in V-ATPase mutants supports this hypothesis. The loss in viability seen in wild-type cells under the same conditions was far less dramatic.

Received for publication September 21, 2006.

Accepted for publication November 8, 2006.

Address correspondence to: Barry Panaretou, Pharmaceutical Science Research Division, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH. E-mail: barry.panaretou{at}kcl.ac.uk

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