We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.
Oxidative stress is known to enhance the frequency of two major types of alterations in the mitochondrial genome of Saccharomyces cerevisiae: point mutations and large deletions resulting in the generation of respiration-deficient petite rhō mutants. We investigated the effect of antimycin A, a well-known agent inducing oxidative stress, on the stability of mtDNA. We show that antimycin enhances exclusively the generation of respiration-deficient petite mutants and this is accompanied by a significant increase in the level of reactive oxygen species (ROS) and in a marked drop of cellular ATP. Whole mitochondrial genome sequencing revealed that mtDNAs of antimycin-induced petite mutants are deleted for most of the wild-type sequence and usually contain one of the active origins of mtDNA replication: ori1, ori2 ori3 or ori5. We show that the frequency of antimycin-induced rhō mutants is significantly elevated in mutants deleted either for the RAD50 or XRS2 gene, both encoding the components of the MRX complex, which is known to be involved in the repair of double strand breaks (DSBs) in DNA. Furthermore, enhanced frequency of rhō mutants in cultures of antimycin-treated cells lacking Rad50 was further increased by the simultaneous absence of the Ogg1 glycosylase, an important enzyme functioning in mtBER. We demonstrate also that rad50Δ and xrs2Δ deletion mutants display a considerable reduction in the frequency of allelic mitochondrial recombination, suggesting that it is the deficiency in homologous recombination which is responsible for enhanced rearrangements of mtDNA in antimycin-treated cells of these mutants. Finally, we show that the generation of large-scale mtDNA deletions induced by antimycin is markedly decreased in a nuc1Δ mutant lacking the activity of the Nuc1 nuclease, an ortholog of the mammalian mitochondrial nucleases EndoG and ExoG. This result indicates that the nuclease plays an important role in processing of oxidative stress-induced lesions in the mitochondrial genome.
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