RESUMEN

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.

Asunto(s)

Quinasa de la Caseína I/metabolismo , Segregación Cromosómica , Proteínas Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Quinasa de la Caseína I/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , ADN de Hongos/genética , Recombinación Homóloga , Meiosis , Mutación , Fosforilación , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejo Sinaptonémico/metabolismo , Cohesinas

RESUMEN

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.

Asunto(s)

Reparación del ADN por Unión de Extremidades/fisiología , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Orden Génico , Genoma Mitocondrial , Autoantígeno Ku , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Genéticos , Tasa de Mutación , Fenotipo , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Transducción de Señal

RESUMEN

The unique segregation of homologs, rather than sister chromatids, at the first meiotic division requires the formation of crossovers (COs) between homologs by meiotic recombination in most species. Crossovers do not form at random along chromosomes. Rather, their formation is carefully controlled, both at the stage of formation of DNA double-strand breaks (DSBs) that can initiate COs and during the repair of these DSBs. Here, we review control of DSB formation and two recently recognized controls of DSB repair: CO homeostasis and CO invariance. Crossover homeostasis maintains a constant number of COs per cell when the total number of DSBs in a cell is experimentally or stochastically reduced. Crossover invariance maintains a constant CO density (COs per kb of DNA) across much of the genome despite strong DSB hotspots in some intervals. These recently uncovered phenomena show that CO control is even more complex than previously suspected.

Asunto(s)

Cromátides/genética , Intercambio Genético , Meiosis , Recombinación Genética , Complejo Sinaptonémico/genética , Animales , Caenorhabditis elegans , Cromátides/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Ratones , Saccharomyces cerevisiae , Schizosaccharomyces , Complejo Sinaptonémico/metabolismo

RESUMEN

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.

Asunto(s)

Centrómero/genética , Heterocromatina/metabolismo , Meiosis/genética , Interferencia de ARN , Recombinación Genética , Proteínas Represoras/metabolismo , Schizosaccharomyces/citología , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/genética , Roturas del ADN de Doble Cadena , Histonas/metabolismo , Lisina/metabolismo , Metiltransferasas/metabolismo , Mutación/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcripción Genética

RESUMEN

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle's high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5' flap structures generated during DNA synthesis. Furthermore, removal of 5' flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity.

Asunto(s)

ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Animales , Reparación del ADN , Endonucleasas de ADN Solapado/deficiencia , Endonucleasas de ADN Solapado/genética , Eliminación de Gen , Masculino , Ratones , Microscopía Fluorescente , Transporte de Proteínas , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.

Asunto(s)

ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mutación , N-Glicosil Hidrolasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Mutación del Sistema de Lectura , Expresión Génica , Genoma Fúngico , Modelos Biológicos , N-Glicosil Hidrolasas/genética , Oxígeno/metabolismo , Mutación Puntual , Recombinación Genética , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/genética , Rayos Ultravioleta

RESUMEN

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.

Asunto(s)

ADN Mitocondrial/genética , Eliminación de Gen , Modelos Genéticos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Saccharomyces cerevisiae/genética , Southern Blotting , Mutación/genética , Reacción en Cadena de la Polimerasa

RESUMEN

The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae. A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified. Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p. Sed1p is a cell surface protein highly expressed in the stationary phase. We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro. Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration. In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability. Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria. Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K. Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p. Our results point towards a role for Sed1p in mitochondrial genome maintenance.

Asunto(s)

Genoma Fúngico , Glicoproteínas de Membrana/fisiología , Mitocondrias/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Secuencia de Bases , ADN Mitocondrial/genética , Técnica del Anticuerpo Fluorescente , Glicoproteínas de Membrana/metabolismo , Mutación Puntual , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos