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Oxidative DNA Damage Causes Mitochondrial Genomic Instability in Saccharomyces cerevisiae

Department of Biochemistry,¹ Graduate Program in Genetic and Molecular Biology, Emory University, Atlanta, Georgia 30322,² Department of Pathology, Yale University, New Haven, Connecticut 06520,³ Department of Biochemistry, Division of Cancer Biology, and Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia 30322⁴

Received 7 January 2005/ Returned for modification 28 February 2005/ Accepted 23 March 2005

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H₂O₂ or utilizing mutants lacking mitochondrial superoxide dismutase (sod2). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1) and oxidative damage resistance (pif1) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H₂O₂. Mitochondrial genomic integrity was substantially compromised in ntg1 pif1 sod2 strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H₂O₂. This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

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