RESUMEN
Cells are constantly confronted with endogenous and exogenous factors that affect their genomes. Eons of evolution have allowed the cellular mechanisms responsible for preserving the genome to adjust for achieving contradictory objectives: to maintain the genome unchanged and to acquire mutations that allow adaptation to environmental changes. One evolutionary mechanism that has been refined for survival is genetic variation. In this review, we describe the mechanisms responsible for two biological processes: genome maintenance and mutation tolerance involved in generations of genetic variations in mitotic cells of both Saccharomyces cerevisiae and Schizosaccharomyces pombe. These processes encompass mechanisms that ensure the fidelity of replication, DNA lesion sensing and DNA damage response pathways, as well as mechanisms that ensure precision in chromosome segregation during cell division. We discuss various factors that may influence genome stability, such as cellular ploidy, the phase of the cell cycle, transcriptional activity of a particular region of DNA, the proficiency of DNA quality control systems, the metabolic stage of the cell and its respiratory potential, and finally potential exposure to endogenous or environmental stress.
Asunto(s)
Inestabilidad Genómica/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Estrés Fisiológico , Daño del ADN/fisiología , Replicación del ADN/genética , Ambiente , Variación Genética , MutaciónRESUMEN
The proteasome plays fundamental roles in the removal of oxidized proteins and in normal degradation of short-lived proteins. Increasing evidence suggests that the proteasome may be an important factor in both oxidative stress response and cellular aging. Moreover, it was recently reported that proteasome inhibition leads to mitochondrial dysfunction. In this study, we have investigated whether proteasome impairment, caused by deletion of UMP1, a gene necessary for the 20S proteasome biogenesis, may influence the stability of the yeast mitochondrial genome. Here we show that an ump1Delta mutant displays enhanced mitochondrial point mutagenesis, measured by the frequency of oligomycin-resistant (Oli(r)) and erythromycin-resistant (Ery(r)) mutants, compared to that of the isogenic wild-type strain. Deletion of UMP1 significantly increases also the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. We show that this mitochondrial mutator phenotype of the ump1Delta strain is considerably reduced in the presence of a plasmid encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS, which was shown previously to counteract oxidative lesion-induced instability of mtDNA. In search of the mechanism underlying the decreased stability of mtDNA in the ump1Delta deletion mutant, we have determined the level of reactive oxygen species (ROS) in the mutant cells and have found that they are exposed to endogenous oxidative stress. Furthermore, we show also that both cellular and intramitochondrial levels of Msh1p are significantly reduced in the mutant cells compared to the wild-type cells. We conclude, therefore, that both an increased ROS production and a markedly decreased level of Msh1p, a protein crucial for the repair of mtDNA, lead in S. cerevisiae cells with impaired proteasome activity to the increased instability of their mitochondrial genome.
Asunto(s)
ADN Mitocondrial/genética , Mitocondrias/genética , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/genética , Antibacterianos/farmacología , Northern Blotting , Western Blotting , Cromosomas Fúngicos/genética , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Fúngica , Eritromicina/farmacología , Genoma Fúngico , Genoma Mitocondrial , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Mutación/genética , Oligomicinas/farmacología , Oxidación-Reducción , Estrés Oxidativo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli(r) mutants in an ogg1Delta strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli(r) mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8(m) recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial genome.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas Fúngicas/fisiología , Inestabilidad Genómica , Estrés Oxidativo/genética , Recombinación Genética , Saccharomyces cerevisiae , ADN Glicosilasas/deficiencia , Enzimas Reparadoras del ADN/deficiencia , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Mitocondrial/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/deficiencia , Proteínas de Unión al ADN , Endodesoxirribonucleasas/deficiencia , Guanina/análogos & derivados , Guanina/metabolismo , Proteínas Mitocondriales , Mutación , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Superóxido Dismutasa/deficienciaRESUMEN
The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3'-to-5' exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3Delta) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3Delta strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation.
Asunto(s)
Mitocondrias/genética , Mitocondrias/metabolismo , Estabilidad del ARN , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Transcripción Genética/genética , Alelos , Sustitución de Aminoácidos/genética , Núcleo Celular/metabolismo , Respiración de la Célula , ARN Helicasas DEAD-box , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Eliminación de Gen , Genoma Fúngico/genética , Intrones/genética , Proteínas Mitocondriales , Fenotipo , ARN Helicasas/genética , ARN de Hongos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Supresión Genética/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.