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1.
Int J Mol Sci ; 22(22)2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34830155

RESUMO

VPS13 proteins are evolutionarily conserved. Mutations in the four human genes (VPS13A-D) encoding VPS13A-D proteins are linked to developmental or neurodegenerative diseases. The relationship between the specific localization of individual VPS13 proteins, their molecular functions, and the pathology of these diseases is unknown. Here we used a yeast model to establish the determinants of Vps13's interaction with the membranes of Golgi apparatus. We analyzed the different phenotypes of the arf1-3 arf2Δ vps13∆ strain, with reduced activity of the Arf1 GTPase, the master regulator of Golgi function and entirely devoid of Vps13. Our analysis led us to propose that Vps13 and Arf1 proteins cooperate at the Golgi apparatus. We showed that Vps13 binds to the Arf1 GTPase through its C-terminal Pleckstrin homology (PH)-like domain. This domain also interacts with phosphoinositol 4,5-bisphosphate as it was bound to liposomes enriched with this lipid. The homologous domain of VPS13A exhibited the same behavior. Furthermore, a fusion of the PH-like domain of Vps13 to green fluorescent protein was localized to Golgi structures in an Arf1-dependent manner. These results suggest that the PH-like domains and Arf1 are determinants of the localization of VPS13 proteins to the Golgi apparatus in yeast and humans.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator 1 de Ribosilação do ADP/genética , Clatrina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Temperatura
2.
J Mol Med (Berl) ; 99(3): 415-423, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33474647

RESUMO

REV3L encodes a catalytic subunit of DNA polymerase zeta (Pol zeta) which is essential for the tolerance of DNA damage by inducing translesion synthesis (TLS). So far, the only Mendelian disease associated with REV3L was Moebius syndrome (3 patients with dominant REV3L mutations causing monoallelic loss-of-function were reported). We describe a homozygous ultra-rare REV3L variant (T2753R) identified with whole exome sequencing in a child without Moebius syndrome but with developmental delay, hypotrophy, and dysmorphic features who was born to healthy parents (heterozygous carriers of the variant). The variant affects the amino acid adjacent to functionally important KKRY motif. By introducing an equivalent mutation (S1192R) into the REV3 gene in yeasts, we showed that, whereas it retained residual function, it caused clear dysfunction of TLS in the nucleus and instability of mitochondrial genetic information. In particular, the mutation increased UV sensitivity measured by cell survival, decreased both the spontaneous (P < 0.005) and UV-induced (P < 0.0001) mutagenesis rates of nuclear DNA and increased the UV-induced mutagenesis rates of mitochondrial DNA (P < 0.0005). We propose that our proband is the first reported case of a REV3L associated disease different from Moebius syndrome both in terms of clinical manifestations and inheritance (autosomal recessive rather than dominant). KEY MESSAGES: First description of a human recessive disorder associated with a REV3L variant. A study in yeast showed that the variant affected the enzymatic function of the protein. In particular, it caused increased UV sensitivity and abnormal mutagenesis rates.


Assuntos
Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Deficiências do Desenvolvimento/genética , Mutação de Sentido Incorreto , Neoplasias Primárias Múltiplas/genética , Síndromes Neoplásicas Hereditárias/genética , Nevo Pigmentado/genética , Mutação Puntual , Neoplasias Cutâneas/genética , Aldose-Cetose Isomerases/genética , Domínio Catalítico/genética , Pré-Escolar , DNA/metabolismo , DNA Fúngico/genética , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/fisiologia , Deficiências do Desenvolvimento/patologia , Feminino , Homozigoto , Humanos , Masculino , Síndrome de Möbius/genética , Modelos Moleculares , Mutagênese/efeitos da radiação , Linhagem , Fenótipo , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Raios Ultravioleta/efeitos adversos , Sequenciamento do Exoma
3.
PLoS One ; 12(7): e0180153, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28678842

RESUMO

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.


Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/fisiologia , Endonucleases Flap/genética , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Acetiltransferases/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/genética , Dano ao DNA , DNA Fúngico/genética , DNA Mitocondrial/genética , Ativação Enzimática , Endonucleases Flap/metabolismo , Técnicas de Inativação de Genes , Instabilidade Genômica , Proteínas de Membrana/metabolismo , Mutagênese , Mutação Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Hum Genet ; 134(9): 951-66, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26077851

RESUMO

Replication of the mitochondrial genome depends on the single DNA polymerase (pol gamma). Mutations in the POLG gene, encoding the catalytic subunit of the human polymerase gamma, have been linked to a wide variety of mitochondrial disorders that show remarkable heterogeneity, with more than 200 sequence variants, often very rare, found in patients. The pathogenicity and dominance status of many such mutations remain, however, unclear. Remarkable structural and functional conservation of human POLG and its S. cerevisiae ortholog (Mip1p) led to the development of many successful yeast models, enabling to study the phenotype of putative pathogenic mutations. In a group of patients with suspicion of mitochondrial pathology, we identified five novel POLG sequence variants, four of which (p.Arg869Ter, p.Gln968Glu, p.Thr1053Argfs*6, and p.Val1106Ala), together with one previously known but uncharacterised variant (p.Arg309Cys), were amenable to modelling in yeast. Familial analysis indicated causal relationship of these variants with disease, consistent with autosomal recessive inheritance. To investigate the effect of these sequence changes on mtDNA replication, we obtained the corresponding yeast mip1 alleles (Arg265Cys, Arg672Ter, Arg770Glu, Thr809Ter, and Val863Ala, respectively) and tested their effect on mitochondrial genome stability and replication fidelity. For three of them (Arg265Cys, Arg672Ter, and Thr809Ter), we observed a strong, partially dominant phenotype of a complete loss of functional mtDNA, whereas the remaining two led to partial mtDNA depletion and significant increase in point mutation frequencies. These results show good correlation with the severity of symptoms observed in patients and allow to establish these variants as pathogenic mutations.


Assuntos
Replicação do DNA , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Saccharomyces cerevisiae/genética , Adolescente , Alelos , Sequência de Aminoácidos , Pré-Escolar , Clonagem Molecular , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Fenótipo , Mutação Puntual , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Free Radic Biol Med ; 82: 73-104, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25640729

RESUMO

The proper functioning of the cell depends on preserving the cellular genome. In yeast cells, a limited number of genes are located on mitochondrial DNA. Although the mechanisms underlying nuclear genome maintenance are well understood, much less is known about the mechanisms that ensure mitochondrial genome stability. Mitochondria influence the stability of the nuclear genome and vice versa. Little is known about the two-way communication and mutual influence of the nuclear and mitochondrial genomes. Although the mitochondrial genome replicates independent of the nuclear genome and is organized by a distinct set of mitochondrial nucleoid proteins, nearly all genome stability mechanisms responsible for maintaining the nuclear genome, such as mismatch repair, base excision repair, and double-strand break repair via homologous recombination or the nonhomologous end-joining pathway, also act to protect mitochondrial DNA. In addition to mitochondria-specific DNA polymerase γ, the polymerases α, η, ζ, and Rev1 have been found in this organelle. A nuclear genome instability phenotype results from a failure of various mitochondrial functions, such as an electron transport chain activity breakdown leading to a decrease in ATP production, a reduction in the mitochondrial membrane potential (ΔΨ), and a block in nucleotide and amino acid biosynthesis. The loss of ΔΨ inhibits the production of iron-sulfur prosthetic groups, which impairs the assembly of Fe-S proteins, including those that mediate DNA transactions; disturbs iron homeostasis; leads to oxidative stress; and perturbs wobble tRNA modification and ribosome assembly, thereby affecting translation and leading to proteotoxic stress. In this review, we present the current knowledge of the mechanisms that govern mitochondrial genome maintenance and demonstrate ways in which the impairment of mitochondrial function can affect nuclear genome stability.


Assuntos
Replicação do DNA/genética , DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Núcleo Celular/genética , Reparo do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Estresse Oxidativo , Saccharomyces cerevisiae/metabolismo
6.
Mutat Res ; 740(1-2): 21-33, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23276591

RESUMO

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 rho 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 rho 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 rho 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.


Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Exonucleases/metabolismo , Instabilidade Genômica , Estresse Oxidativo/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Antimicina A/farmacologia , DNA Glicosilases , DNA Fúngico/metabolismo , Proteínas Mitocondriais/genética , Mutação , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética
7.
Biochim Biophys Acta ; 1797(6-7): 1086-98, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20056105

RESUMO

As a legacy of their endosymbiotic eubacterial origin, mitochondria possess a residual genome, encoding only a few proteins and dependent on a variety of factors encoded by the nuclear genome for its maintenance and expression. As a facultative anaerobe with well understood genetics and molecular biology, Saccharomyces cerevisiae is the model system of choice for studying nucleo-mitochondrial genetic interactions. Maintenance of the mitochondrial genome is controlled by a set of nuclear-coded factors forming intricately interconnected circuits responsible for replication, recombination, repair and transmission to buds. Expression of the yeast mitochondrial genome is regulated mostly at the post-transcriptional level, and involves many general and gene-specific factors regulating splicing, RNA processing and stability and translation. A very interesting aspect of the yeast mitochondrial system is the relationship between genome maintenance and gene expression. Deletions of genes involved in many different aspects of mitochondrial gene expression, notably translation, result in an irreversible loss of functional mtDNA. The mitochondrial genetic system viewed from the systems biology perspective is therefore very fragile and lacks robustness compared to the remaining systems of the cell. This lack of robustness could be a legacy of the reductive evolution of the mitochondrial genome, but explanations involving selective advantages of increased evolvability have also been postulated.


Assuntos
Genoma Fúngico , Genoma Mitocondrial , Saccharomyces cerevisiae/genética , Reparo do DNA , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Evolução Molecular , Regulação Fúngica da Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Genéticos , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mitocondrial , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Biologia de Sistemas
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