Oxidative DNA Damage Causes Mitochondrial Genomic Instability in Saccharomyces cerevisiae

Abstract

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 H 2 O 2 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 H 2 O 2 . 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 H 2 O 2 . 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.

Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

“…To test the role of mPif1 in chromosome healing, we plan to make use of an elegant system developed by Murnane and colleagues, in which a telomere can be cleaved via a unique I-SceI site integrated into the subtelomeric DNA of one chromosome (39,64). Since deletion of ScPIF1 leads to an increase in mitochondrial DNA point mutations, particularly after oxidative damage (21,51,52,67), it is also possible mPif1 plays a nonessential role in mitochondrial genome stability. Although Scrrm3⌬ cells do not exhibit changes in GCR or mitochondrial genome stability, these cells do exhibit genetic instability, presumably due to replication fork pausing at specific chromosomal loci, such as the ribosomal DNA locus (31,32,45,59,68,69).…”

“…Interestingly, ScPif1 (but not Rrm3) colocalizes almost exclusively with Rad52 in nuclei and is recruited to discrete foci after DNA damage (71). ScPif1 and Rrm3 also play roles in the mitochondria, where they contribute to mitochondrial genome stability (21,37,51,52,67,70). Finally, both ScPif1 and Pfh1 interact genetically with the Dna2 helicase and cooperate to ensure the correct processing of Okazaki fragments during DNA replication (17,58).…”

Mitochondrial Reactive Oxygen Species. Contribution to Oxidative Stress and Interorganellar Signaling

“…Oxidative Damage to mtDNA Hydroxyl radicals are highly reactive and can damage nuclear and mitochondrial DNA, which cells try to repair (Roldan-Arjona et al, 2000; Doudican et al, 2005). Accumulated damage to mtDNA, which can be caused by mtROS over the course of the lives of animals, causes decreased mitochondrial function and can contribute to aging and diseases (Allen, 1996;Raha and Robinson, 2000;Trifunovic et al, 2004;Wallace, 2005).…”

Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis

“…The 8-oxodG adduct is formed by the reaction of the hydroxyl radical with the DNA guanine base and is a promutagenic lesion that mispairs with adenine, leading to GC-to-TA transversion. Oxidative DNA damage induced by ROS could potentially be a major source of mitochondrial genomic instability, leading to respiratory chain dysfunction (4,5).…”