RESUMEN
The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.
Asunto(s)
ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Mitocondrias/enzimología , Sustitución de Aminoácidos , Secuencia de Bases , Línea Celular , ADN Polimerasa gamma , ADN Mitocondrial/química , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Partículas Submitocóndricas/enzimología , Transfección , Repeticiones de TrinucleótidosRESUMEN
In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens. Brief incursions into systems of other organisms have also been achieved when they provide new information.S. cerevisiae and H. sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms. However, recent studies suggest that mitochondrial genome of S. cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular. Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident. As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast. Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously. Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products. Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections. These links are presented on a map. Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication.
Asunto(s)
Replicación del ADN , ADN Mitocondrial/genética , Saccharomyces cerevisiae/genética , HumanosRESUMEN
The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.
Asunto(s)
ADN Mitocondrial , ADN Polimerasa Dirigida por ADN/genética , Mitocondrias/enzimología , Repeticiones de Trinucleótidos , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN Polimerasa gamma , ADN Complementario , Humanos , Datos de Secuencia MolecularRESUMEN
The yeast two-hybrid system is a genetic method that detects protein-protein interactions. One application is the detection by library screening of new interactors of a protein of known function. In the August issue of Nature Genetics, Fromont-Racine et al. showed for the first time that the construction of the protein interaction map of a complex pathway, such as that of the mRNA splicing machinery, is now possible, because of the combination of recent technical improvements elaborated in several laboratories. With a yeast cell mating procedure that increases screen efficiency, they used their complex yeast genomic library of 5 x 10(6) clones to test 700 x 10(6) interactions against 15 proteins. They identified and classified 170 potential interactors, including approximately 70 proteins of previously unknown function. More than 25% of the interactors are probably biologically relevant. The achievements of Fromont-Racine et al. have opened the way to the systematic analysis of the protein interaction networks of the 6,000 open reading frames-yeast proteome. This task requires, however, automation of the library screens and creation of a two-hybrid library database.
Asunto(s)
Saccharomyces cerevisiae/genética , Proteínas Fúngicas/genética , Técnicas Genéticas , Genoma Fúngico , Biblioteca Genómica , Hibridación Genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85,779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.
Asunto(s)
ADN de Hongos/genética , ADN Mitocondrial/genética , Saccharomyces cerevisiae/genética , Composición de Base , Secuencia de Bases , Citosina/análisis , Eliminación de Gen , Genoma Fúngico , Guanosina/análisis , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Polimorfismo Genético , Alineación de SecuenciaRESUMEN
We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases gamma (Pol gamma). Multiple amino acid sequence alignments with Pol gamma from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Melanoma/genética , Mitocondrias/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , ADN Polimerasa Dirigida por ADN/clasificación , Drosophila melanogaster , Exonucleasas/genética , Biblioteca de Genes , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Pichia , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae , Schizosaccharomyces , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
A multicopy suppressor gene which rescues the temperature-sensitive growth defect of Saccharomyces cerevisiae mutants in the mitochondrial DNA (mtDNA) polymerase-encoding MIP1 gene has been isolated and identified as the RNR1 gene. This gene, whose transcript is cell cycle-regulated and mainly expressed at the G1 to S phase transition, encodes the large subunit of ribonucleotide reductase. This enzyme catalyses a limiting step in the production of deoxynucleotides needed for DNA synthesis. The presence of a high copy number of the RNR1 gene also decreases the accumulation of rho- mutants observed in diploids that harbour a single copy of the MIP1 gene. In cell cycle-synchronised cells, the presence of a high copy number of RNR1 does not modify its cell cycle transcription regulation and increases its transcript level by a factor of 10 throughout the cell cycle. Our results show that an increased supply of dNTPs in mitochondria can stimulate the mtDNA polymerase activity and indicate that the dNTP concentration may be rate limiting for the replication of mtDNA.