RESUMO
Most eukaryotes possess a mitochondrial genome, called mtDNA. In animals and fungi, the replication of mtDNA is entrusted by the DNA polymerase γ, or Pol γ. The yeast Pol γ is composed only of a catalytic subunit encoded by MIP1. In humans, Pol γ is a heterotrimer composed of a catalytic subunit homolog to Mip1, encoded by POLG, and two accessory subunits. In the last 25 years, more than 300 pathological mutations in POLG have been identified as the cause of several mitochondrial diseases, called POLG-related disorders, which are characterized by multiple mtDNA deletions and/or depletion in affected tissues. In this review, at first, we summarize the biochemical properties of yeast Mip1, and how mutations, especially those introduced recently in the N-terminal and C-terminal regions of the enzyme, affect the in vitro activity of the enzyme and the in vivo phenotype connected to the mtDNA stability and to the mtDNA extended and point mutability. Then, we focus on the use of yeast harboring Mip1 mutations equivalent to the human ones to confirm their pathogenicity, identify the phenotypic defects caused by these mutations, and find both mechanisms and molecular compounds able to rescue the detrimental phenotype. A closing chapter will be dedicated to other polymerases found in yeast mitochondria, namely Pol ζ, Rev1 and Pol η, and to their genetic interactions with Mip1 necessary to maintain mtDNA stability and to avoid the accumulation of spontaneous or induced point mutations.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Humanos , DNA Polimerase gama/genética , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Mitocondrial/genética , Mutação , Replicação do DNA/genéticaRESUMO
(1) Background: Hepatitis C virus (HCV) infection is endemic in Egypt, with the highest prevalence rate worldwide. Sofosbuvir (SOF) is a nucleos(t)ide analog that specifically inhibits HCV replication. This study aimed to explore the possible effects of the therapeutic dose of SOF on the mitochondrial biogenesis and functions of the liver, muscle, and ovarian tissues of young normal female rats. (2) Methods: This study was conducted on 20 female Wistar rats, classified into two groups, the control group and the exposed group; the latter was orally supplemented with 4 mg/kg/day of SOF for 3 months. (3) Results: The exposure to SOF impairs mitochondrial biogenesis via mitochondrial DNA copy number decline and suppressed mitochondrial biogenesis-regulated parameters at mRNA and protein levels. Also, SOF suppresses the DNA polymerase γ (POLG) expression, citrate synthase activity, and mitochondrial NADH dehydrogenase subunit-5 (ND5) content, which impairs mitochondrial functions. SOF increased lipid peroxidation and oxidative DNA damage markers and decreased tissue expression of nuclear factor erythroid 2-related factor 2 (Nfe2l2). (4) Conclusions: The present findings demonstrate the adverse effects of SOF on mitochondrial biogenesis and function in different tissues of young female rats, which mostly appeared in ovarian tissues.
Assuntos
Hepatite C Crônica , Hepatite C , Feminino , Ratos , Animais , Antivirais , Hepatite C Crônica/tratamento farmacológico , Biogênese de Organelas , Resultado do Tratamento , Ratos Wistar , Sofosbuvir/uso terapêutico , Hepatite C/tratamento farmacológico , Hepacivirus/genética , Quimioterapia Combinada , GenótipoRESUMO
Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex-forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.
Assuntos
DNA Polimerase gama/metabolismo , DNA Mitocondrial/metabolismo , Quadruplex G , Biocatálise , Replicação do DNA/efeitos da radiação , Humanos , Mitocôndrias/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Regiões Promotoras Genéticas , Especificidade por Substrato , Raios UltravioletaRESUMO
We establish the genotype-phenotype correlations for the complete spectrum of POLG syndromes by refining our previously described protocol for mapping pathogenic mutations in the human POLG gene to functional clusters in the catalytic core of the mitochondrial replicase, Pol γ (1). We assigned 136 mutations to five clusters and identify segments of primary sequence that can be used to delimit the boundaries of each cluster. We report that compound heterozygotes with two mutations from different clusters manifested more severe, earlier-onset POLG syndromes, whereas two mutations from the same cluster are less common and generally are associated with less severe, later onset POLG syndromes. We also show that specific cluster combinations are more severe than others and have a higher likelihood to manifest at an earlier age. Our clustering method provides a powerful tool to predict the pathogenic potential and predicted disease phenotype of novel variants and mutations in POLG, the most common nuclear gene underlying mitochondrial disorders. We propose that such a prediction tool would be useful for routine diagnostics for mitochondrial disorders. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
Assuntos
DNA Polimerase Dirigida por DNA/química , Doenças Mitocondriais/genética , Mutação , Fenótipo , Sequência de Aminoácidos , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Genótipo , Heterozigoto , Humanos , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.
Assuntos
Adenosina/química , Antivirais/química , DNA Polimerase Dirigida por DNA/química , Inibidores da Síntese de Ácido Nucleico/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA/química , Vírus Sinciciais Respiratórios/enzimologia , Vírus Sinciciais Respiratórios/fisiologia , Adenosina/síntese química , Adenosina/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Compostos Aza/química , DNA Polimerase Dirigida por DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA/metabolismo , RNA Mitocondrial , RNA Polimerase Dependente de RNA/metabolismo , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Nucleoside reverse transcriptase inhibitors (NRTIs)/nucleotide reverse transcriptase inhibitors are key components of combination antiretroviral therapy for HIV infection. First-generation NRTIs are associated with mitochondrial toxicity in patients, mainly due to inhibition of human DNA polymerase γ (hDNA polγ) that manifests as adverse events such as lipodystrophy, lactic acidosis, myopathy, cardiomyopathy, or nephropathy in patients. In chronic nonclinical studies in rodents and nonrodents, eukaryotic (host) mitochondrial toxicity manifests as some drug-specific toxicities similar to human toxicity. BMS-986001, a novel thymidine analog with minimal hDNA polγ inhibition, has demonstrated antiretroviral activity in early clinical studies. The primary toxicity of BMS-986001 in rats and monkeys is bone marrow dyserythropoiesis with associated decreases in red blood cell mass. Additionally, at high doses, severe platelet reductions accompanied by cutaneous petechiae began during weeks 8 and 11 in 3 of 60 monkeys in chronic toxicity studies. In a 6-month study, platelet reductions required euthanasia of the 2 affected monkeys (300 mg/kg/d) at week 14, but with dose reduction (200 mg/kg/d) remaining monkeys had no platelet changes. One affected monkey (200 mg/kg/d) in a 9-month study completed dosing and its platelet counts recovered during a 1-month recovery. Formation of platelet-bound immunoglobulin in the presence of BMS-986001, together with rapid and complete platelet recovery in the absence of BMS-986001, suggested that platelet decreases in monkeys may be immune mediated. No findings indicative of mitochondrial toxicity were observed in rats or monkeys given BMS-986001, suggesting an improved safety profile compared to marketed NRTI or tenofovir disoproxil fumarate.
Assuntos
Anemia Macrocítica/induzido quimicamente , Fármacos Anti-HIV/efeitos adversos , Drogas em Investigação/efeitos adversos , Púrpura Trombocitopênica/induzido quimicamente , Inibidores da Transcriptase Reversa/efeitos adversos , Timidina/análogos & derivados , Anemia Macrocítica/sangue , Anemia Macrocítica/metabolismo , Anemia Macrocítica/patologia , Animais , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/metabolismo , Biotransformação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/administração & dosagem , Drogas em Investigação/metabolismo , Eritropoese/efeitos dos fármacos , Feminino , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Meia-Vida , Macaca fascicularis , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Púrpura Trombocitopênica/imunologia , Púrpura Trombocitopênica/metabolismo , Púrpura Trombocitopênica/patologia , Distribuição Aleatória , Ratos Sprague-Dawley , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/sangue , Inibidores da Transcriptase Reversa/metabolismo , Análise de Sobrevida , Timidina/administração & dosagem , Timidina/efeitos adversos , Timidina/sangue , Timidina/metabolismo , Testes de Toxicidade Crônica , ToxicocinéticaRESUMO
Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus Metarhizium brunneum is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of mip1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of mip1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the M. brunneum Mip1 and its counterparts from Saccharomyces cerevisiae and higher eukaryotes are discussed.
RESUMO
Mutations in the catalytic domain of mitochondrial DNA polymerase γ (POLγ) cause a broad spectrum of clinical conditions. POLγ mutations impair mitochondrial DNA replication, thereby causing deletions and/or depletion of mitochondrial DNA, which in turn impair biogenesis of the oxidative phosphorylation system. We here identify a patient with a homozygous p.F907I mutation in POLγ, manifesting a severe clinical phenotype with developmental arrest and rapid loss of skills from 18 months of age. Magnetic resonance imaging of the brain revealed extensive white matter abnormalities, Southern blot of muscle mtDNA demonstrated depletion of mtDNA and the patient deceased at 23 months of age. Interestingly, the p.F907I mutation does not affect POLγ activity on single-stranded DNA or its proofreading activity. Instead, the mutation affects unwinding of parental double-stranded DNA at the replication fork, impairing the ability of the POLγ to support leading-strand DNA synthesis with the TWINKLE helicase. Our results thus reveal a novel pathogenic mechanism for POLγ-related diseases.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA Polimerase gama/genética , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação , Humanos , LactenteRESUMO
One of the most proliferative periods for T cells occurs during their development in the thymus. Increased DNA replication can result in increased DNA mutations in the nuclear genome, but also in mitochondrial genomes. A high frequency of mitochondrial DNA mutations can lead to abnormal mitochondrial function and have negative implications on human health. Furthermore, aging is accompanied by an increase in such mutations through oxidative damage and replication errors. Increased mitochondrial DNA mutations cause loss of mitochondrial protein function, and decrease energy production, substrates, and metabolites. Here we have evaluated the effect of increased mitochondrial DNA mutations on T cell development in the thymus. Using mice carrying a mutant mitochondrial DNA polymerase γ (PolG) that causes increased mitochondrial DNA mutations, we show that high fidelity replication of mitochondrial DNA is pivotal for proper T cell development. Reducing the fidelity of mitochondrial DNA replication results in a premature age-dependent reduction in the total number of CD4/CD8 double negative and double positive thymocytes. Analysis of mitochondrial density in thymocyte subpopulations suggests that this may be due to reduced proliferation in specific double negative stages. Taken together, this work suggests that T cell development is regulated by the ability of mitochondria to faithfully replicate their DNA.
Assuntos
DNA Mitocondrial , Timócitos , Humanos , Camundongos , Animais , Timócitos/metabolismo , DNA Mitocondrial/genética , Diferenciação Celular , Timo/metabolismo , Mitocôndrias/genéticaRESUMO
The manipulation of mitochondrial DNA (mtDNA) copy number in cultured cells, using substances that interfere with DNA replication, is a useful tool to investigate various aspects of mtDNA maintenance. Here we describe the use of 2',3'-dideoxycytidine (ddC) to induce a reversible reduction of mtDNA copy number in human primary fibroblasts and human embryonic kidney (HEK293) cells. Once the application of ddC is stopped, cells depleted for mtDNA attempt to recover normal mtDNA copy numbers. The dynamics of repopulation of mtDNA provide a valuable measure for the enzymatic activity of the mtDNA replication machinery.
Assuntos
DNA Mitocondrial , Zalcitabina , Humanos , Zalcitabina/farmacologia , DNA Mitocondrial/genética , Células HEK293 , Mitocôndrias/genética , Células Cultivadas , Replicação do DNARESUMO
In line with recent OECD activities on the use of AOPs in developing Integrated Approaches to Testing and Assessment (IATAs), it is expected that systematic mapping of AOPs leading to systemic toxicity may provide a mechanistic framework for the development and implementation of mechanism-based in vitro endpoints. These may form part of an integrated testing strategy to reduce the need for repeated dose toxicity studies. Focusing on kidney and in particular the proximal tubule epithelium as a key target site of chemical-induced injury, the overall aim of this work is to contribute to building a network of AOPs leading to nephrotoxicity. Current mechanistic understanding of kidney injury initiated by 1) inhibition of mitochondrial DNA polymerase γ (mtDNA Polγ), 2) receptor mediated endocytosis and lysosomal overload, and 3) covalent protein binding, which all present fairly well established, common mechanisms by which certain chemicals or drugs may cause nephrotoxicity, is presented and systematically captured in a formal description of AOPs in line with the OECD AOP development programme and in accordance with the harmonized terminology provided by the Collaborative Adverse Outcome Pathway Wiki. The relative level of confidence in the established AOPs is assessed based on evolved Bradford-Hill weight of evidence considerations of biological plausibility, essentiality and empirical support (temporal and dose-response concordance).
RESUMO
Mutations in the mitochondrial DNA polymerase gamma catalytic subunit (POLγA) compromise the stability of mitochondrial DNA (mtDNA) by leading to mutations, deletions and depletions in mtDNA. Patients with mutations in POLγA often differ remarkably in disease severity and age of onset. In this work we have studied the functional consequence of POLγA mutations in a patient with an uncommon and a very severe disease phenotype characterized by prenatal onset with intrauterine growth restriction, lactic acidosis from birth, encephalopathy, hepatopathy, myopathy, and early death. Muscle biopsy identified scattered COX-deficient muscle fibers, respiratory chain dysfunction and mtDNA depletion. We identified a novel POLγA mutation (p.His1134Tyr) in trans with the previously identified p.Thr251Ile/Pro587Leu double mutant. Biochemical characterization of the purified recombinant POLγA variants showed that the p.His1134Tyr mutation caused severe polymerase dysfunction. The p.Thr251Ile/Pro587Leu mutation caused reduced polymerase function in conditions of low dNTP concentration that mimic postmitotic tissues. Critically, when p.His1134Tyr and p.Thr251Ile/Pro587Leu were combined under these conditions, mtDNA replication was severely diminished and featured prominent stalling. Our data provide a molecular explanation for the patient´s mtDNA depletion and clinical features, particularly in tissues such as brain and muscle that have low dNTP concentration.
Assuntos
DNA Polimerase gama/genética , Encefalomiopatias Mitocondriais/genética , Mutação/genética , Replicação do DNA , DNA Mitocondrial , Humanos , Recém-Nascido , Masculino , FenótipoRESUMO
The mitochondrial single-stranded DNA-binding protein (mtSSB) regulates the function of the mitochondrial DNA (mtDNA) replisome. In vitro, mtSSB stimulates the activity of enzymatic components of the replisome, namely mtDNA helicase and DNA polymerase gamma (Pol γ). We have demonstrated that the stimulatory properties of mtSSB result from its ability to organize the single-stranded DNA template in a specific manner. Here we present methods employing electron microscopy and enzymatic assays to characterize and classify the mtSSB-DNA complexes and their effects on the activity of Pol γ.
Assuntos
DNA Polimerase gama/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/metabolismo , DNA de Cadeia Simples/química , Ensaios Enzimáticos , Humanos , Microscopia Eletrônica , Conformação Molecular , Nucleoproteínas/químicaRESUMO
Human DNA polymerase γ (POLG) is a mitochondria-specific replicative DNA polymerase consisting of a single catalytic subunit, POLGα, and a dimeric accessory subunit, POLGß. To gain a deeper understanding of the role of POLGß, we knocked out this protein in cultured human cybrid cells and established numerous knockout clones. POLGß-knockout clones presented a clear phenotype of mitochondrial DNA loss, indicating that POLGß is necessary for mitochondrial DNA replication. Moreover, POLGß-knockout cells showed a severe decrease in POLGα levels and acute suppression of POLGß expression efficiently down-regulated POLGα levels. These results suggest that, in addition to its role as the processivity factor of POLG, POLGß acts as a POLGα stabilizer, an important role for POLGß in mitochondrial DNA maintenance.
Assuntos
DNA Polimerase gama/metabolismo , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mitocôndrias/genética , DNA Polimerase gama/química , DNA Polimerase gama/genética , DNA Mitocondrial/química , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Estabilidade Enzimática , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Células HeLa , Humanos , FenótipoRESUMO
Lake Chaohu, one of the five largest freshwater lakes in China, has been suffering from severe cyanobacterial blooms in the summer for many years. Cyanophages, the viruses that specifically infect cyanobacteria, play a key role in modulating cyanobacterial population, and thus regulate the emergence and decline of cyanobacterial blooms. Here we report a long-tailed cyanophage isolated from Lake Chaohu, termed Mic1, which specifically infects the cyanobacterium Microcystis aeruginosa. Mic1 has an icosahedral head of 88 nm in diameter and a long flexible tail of 400 nm. It possesses a circular genome of 92,627 bp, which contains 98 putative open reading frames. Genome sequence analysis enabled us to define a novel terminase large subunit that consists of two types of intein, indicating that the genome packaging of Mic1 is under fine control via posttranslational maturation of the terminase. Moreover, phylogenetic analysis suggested Mic1 and mitochondria share a common evolutionary origin of DNA polymerase γ gene. All together, these findings provided a start-point for investigating the co-evolution of cyanophages and its cyanobacterial hosts.
RESUMO
Mitochondrial DNA (mtDNA), which is essential for mitochondrial and cell function, is replicated and transcribed in the organelle by proteins that are entirely coded in the nucleus. Replication of mtDNA is challenged not only by threats related to the replication machinery and orchestration of DNA synthesis, but also by factors linked to the peculiarity of this genome. Indeed the architecture, organization, copy number, and location of mtDNA, which are markedly distinct from the nuclear genome, require ad hoc and complex regulation to ensure coordinated replication. As a consequence sub-optimal mtDNA replication, which results from compromised regulation of these factors, is generally associated with mitochondrial dysfunction and disease. Mitochondrial DNA replication should be considered in the context of the organelle and the whole cell, and not just a single genome or a single replication event. Major threats to mtDNA replication are linked to its dependence on both mitochondrial and nuclear factors, which require exquisite coordination of these crucial subcellular compartments. Moreover, regulation of replication events deals with a dynamic population of multiple mtDNA molecules rather than with a fixed number of genome copies, as it is the case for nuclear DNA. Importantly, the mechanistic aspects of mtDNA replication are still debated. We describe here major challenges for human mtDNA replication, the mechanistic aspects of the process that are to a large extent original, and their consequences on disease.
Assuntos
Núcleo Celular/genética , Replicação do DNA , DNA Mitocondrial/genética , Mitocôndrias/genética , Estresse Oxidativo , Genoma Mitocondrial , Humanos , Mitocôndrias/metabolismoRESUMO
Peripheral Nervous System (PNS) neurotoxicity caused by cancer drugs hinders attainment of chemotherapy goals. Due to leakiness of the blood nerve barrier, circulating chemotherapeutic drugs reach PNS neurons and adversely affect their function. Chemotherapeutic drugs are designed to target dividing cancer cells and mechanisms underlying their toxicity in postmitotic neurons remain to be fully clarified. The objective of this work was to elucidate progression of events triggered by antimitotic drugs in postmitotic neurons. For proof of mechanism study, we chose cytarabine (ara-C), an antimetabolite used in treatment of hematological cancers. Ara-C is a cytosine analog that terminates DNA synthesis. To investigate how ara-C affects postmitotic neurons, which replicate mitochondrial but not genomic DNA, we adapted a model of Dorsal Root Ganglion (DRG) neurons. We showed that DNA polymerase γ, which is responsible for mtDNA synthesis, is inhibited by ara-C and that sublethal ara-C exposure of DRG neurons leads to reduction in mtDNA content, ROS generation, oxidative mtDNA damage formation, compromised mitochondrial respiration and diminution of NADPH and GSH stores, as well as, activation of the DNA damage response. Hence, it is plausible that in ara-C exposed DRG neurons, ROS amplified by the high mitochondrial content shifts from physiologic to pathologic levels signaling stress to the nucleus. Combined, the findings suggest that ara-C neurotoxicity in DRG neurons originates in mitochondria and that continuous mtDNA synthesis and reliance on oxidative phosphorylation for energy needs sensitize the highly metabolic neurons to injury by mtDNA synthesis terminating cancer drugs.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Citarabina/toxicidade , DNA Mitocondrial/metabolismo , Gânglios Espinais/patologia , Mitocôndrias/patologia , Síndromes Neurotóxicas , Animais , Células Cultivadas , Dano ao DNA , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transdução de SinaisRESUMO
Deamination of adenine can occur spontaneously under physiological conditions to generate the highly mutagenic lesion, deoxyinosine (hypoxanthine deoxyribonucleotide, dI). In DNA, dI preferably pairs with cytosine rather than thymine and results in A:T to G:C transition mutations after DNA replication. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli, dI repair is initiated by endonuclease V (endo V; nfi gene product) nicking but a complete repair mechanism has yet to be elucidated. Using in vitro minimum component reconstitution assays, we previously showed that endo V, DNA polymerase I (pol I), and E. coli DNA ligase were sufficient to repair this dI lesions efficiently and that the 3'-5' exonuclease of pol I is essential. Here we employed a phagemid-based T-I substrate mimicking adenine deamination product to demonstrate pol I proofreading exonuclease is required by the endo V repair pathway both in vitro and in vivo. In vivo we found that the repair level of an nfi mutant (11%) was almost 8-fold lower than the wild type (87%). while the polA-D424A strain, a pol I mutant defective in 3'-5' exonuclease, showed a high repair level similar to wild type (both more than 80%). Using additional C-C mismatch as strand discrimination marker we found that the high level of dI removal in polA-D424A was due to strand loss (more than 60%) associated with incomplete repair. Thus, pol I proofreading exonuclease is the major function responsible for dI lesion removal after endoV nicking both in vitro and in vivo. Finally, using MALDI-TOF to analyze single-nucleotide extension product we show that the pol I proofreading exonuclease excises only 2-nt 5' upstream of endo V incision site further honing the role of pol I in the endoV dI dependent repair pathway.
Assuntos
Dano ao DNA , DNA Polimerase I/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Inosina/análogos & derivados , DNA/metabolismo , DNA Ligases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Inosina/metabolismoRESUMO
Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) ß. Pol ß was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol ß. Mitochondria from pol ß-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol ß-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol ß and thus pol ß plays a role in preserving mitochondrial genome stability.
Assuntos
Dano ao DNA , DNA Polimerase beta/metabolismo , Reparo do DNA , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Animais , DNA Polimerase beta/genética , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Estresse Oxidativo/efeitos dos fármacos , Superóxidos/análise , Superóxidos/metabolismoRESUMO
BACKGROUND: The recent genome-wide association studies (GWAS) of Type 2 Diabetes (T2D) have identified the pancreatic ß-cell as the culprit in the pathogenesis of the disease. Mitochondrial metabolism plays a crucial role in the processes controlling release of insulin and ß-cell mass. This notion implies that mechanisms controlling mitochondrial function have the potential to play a decisive pathogenetic role in T2D. SCOPE OF THE REVIEW: This article reviews studies demonstrating that there is indeed mitochondrial dysfunction in islets in T2D, and that GWAS have identified a variant in the gene encoding transcription factor B1 mitochondrial (TFB1M), predisposing to T2D due to mitochondrial dysfunction and impaired insulin secretion. Mechanistic studies of the nature of this pathogenetic link, as well as of other mitochondrial transcription factors, are described. MAJOR CONCLUSIONS: Based on this, it is argued that transcription and translation in mitochondria are critical processes determining mitochondrial function in ß-cells in health and disease.