RESUMO
Mitochondria are dynamic organelles that fuse and divide. These changes alter the number and distribution of mitochondrial structures throughout the cell in response to developmental and metabolic cues. We have demonstrated that mitochondrial fission is essential to the maintenance of mitochondrial DNA (mtDNA) under changing metabolic conditions in wild-type Saccharomyces cerevisiae. While increased loss of mtDNA integrity has been demonstrated for dnm1-∆ fission mutants after growth in a non-fermentable carbon source, we demonstrate that growth of yeast in different carbon sources affects the frequency of mtDNA loss, even when the carbon sources are fermentable. In addition, we demonstrate that the impact of fission on mtDNA maintenance during growth in different carbon sources is neither mediated by retrograde signaling nor mitophagy. Instead, we demonstrate that mitochondrial distribution and mtDNA maintenance phenotypes conferred by loss of Dnm1p are suppressed by the loss of Sod2p, the mitochondrial matrix superoxide dismutase.
Assuntos
Genoma Mitocondrial , Instabilidade Genômica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Carbono/metabolismo , Respiração Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Mitocondrial , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Deleção de Genes , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Rafinose/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de SequênciaRESUMO
Loss of Abf2p, an abundant mitochondrial nucleoid-associated protein, results in increased mitochondrial frameshifts and direct-repeat mediated deletions but has no effect on the rate of mitochondrial point mutations. The instability of repeated sequences in this strain may be linked to the loss of mitochondrial DNA in abf2-Delta strains.
Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA/deficiência , Genoma Mitocondrial/genética , Instabilidade Genômica , Sequências Repetitivas de Ácido Nucleico/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/deficiência , Aerobiose/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Instabilidade Genômica/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Mutação/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions.
Assuntos
DNA Mitocondrial/genética , Deleção de Genes , Modelos Genéticos , Sequências Repetitivas de Ácido Nucleico/genética , Saccharomyces cerevisiae/genética , Southern Blotting , Mutação/genética , Reação em Cadeia da PolimeraseRESUMO
Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.
Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Ordem dos Genes , Genoma Mitocondrial , Autoantígeno Ku , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Genéticos , Taxa de Mutação , Fenótipo , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Transdução de SinaisRESUMO
Microsatellites, or simple repetitive sequences, are abundant in eukaryotic genomes and in the mitochondrial genome of Saccharomyces cerevisiae. These sequences alter at rates significantly higher than non-repetitive sequences of comparable size. The stability of a mitochondrial microsatellite is nearly 100-fold greater in diploid yeast cells than in isogenic haploid cells. We were able to demonstrate that this effect is likely due to ploidy alone, rather than mating-type-specific gene expression. In addition, we demonstrated that amino acid starvation affects the organization of the mitochondrial DNA and its segregation into the bud. We also tested the effect of amino acid starvation on the copy number and the mutation rate of mitochondrial DNA in both haploid and diploid yeast cells. Yeast cells grown in rich medium have a lower mitochondrial DNA content than cells starved for amino acids and have a correspondingly higher mutation rate for both frameshift mutations and point mutations in mitochondrial DNA. These effects appear to be dependent on the mitochondrial nucleoid-associated protein Ilv5p.