RESUMO
Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.
Assuntos
Mitocôndrias , Recombinação Genética , Animais , Mitocôndrias/genética , Mitocôndrias/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Reparo do DNA , Replicação do DNA/genética , Mamíferos/genéticaRESUMO
Molecular functions of many human proteins remain unstudied, despite the demonstrated association with diseases or pivotal molecular structures, such as mitochondrial DNA (mtDNA). This small genome is crucial for the proper functioning of mitochondria, the energy-converting organelles. In mammals, mtDNA is arranged into macromolecular complexes called nucleoids that serve as functional stations for its maintenance and expression. Here, we aimed to explore an uncharacterized protein C17orf80, which was previously detected close to the nucleoid components by proximity labelling mass spectrometry. To investigate the subcellular localization and function of C17orf80, we took advantage of immunofluorescence microscopy, interaction proteomics and several biochemical assays. We demonstrate that C17orf80 is a mitochondrial membrane-associated protein that interacts with nucleoids even when mtDNA replication is inhibited. In addition, we show that C17orf80 is not essential for mtDNA maintenance and mitochondrial gene expression in cultured human cells. These results provide a basis for uncovering the molecular function of C17orf80 and the nature of its association with nucleoids, possibly leading to new insights about mtDNA and its expression.
Assuntos
Mitocôndrias , Proteínas Mitocondriais , Animais , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Replicação do DNA , Mamíferos/metabolismoRESUMO
Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.
Assuntos
DNA Mitocondrial , Quadruplex G , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Replicação do DNA/genéticaRESUMO
Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin OH and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.
Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I , Animais , Replicação do DNA/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Mitocondrial/genética , Mamíferos/genética , Mitocôndrias/metabolismo , Origem de ReplicaçãoRESUMO
In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.
Assuntos
DNA Polimerase gama/genética , Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA Polimerase gama/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Heterozigoto , Humanos , MutaçãoRESUMO
Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological and behavioural differences. Each species has travelled its own evolutionary trajectory, influenced by random drift and driven by various types of natural selection, making the association of genetic differences between the species with the phenotypic differences extremely complex to dissect. In the present study, we have used an in vitro model to analyse in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, the arctic/subarctic mountain hare (Lepus timidus Linnaeus) and the temperate steppe-climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1623 genes, manifesting in differences in cell growth, cell cycle control, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest that speciation acts on fundamental cellular processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.
Assuntos
Lebres , Lagomorpha , Animais , Lebres/genética , Lagomorpha/genética , Evolução Biológica , Mamíferos , Regiões ÁrticasRESUMO
Mutations in the mitochondrial inner membrane ATPase ATAD3A result in neurological syndromes in humans. In mice, the ubiquitous disruption of Atad3 (also known as Atad3a) was embryonic lethal, but a skeletal muscle-specific conditional knockout (KO) was viable. At birth, ATAD3 muscle KO mice had normal weight, but from 2â months onwards they showed progressive motor-impaired coordination and weakness. Loss of ATAD3 caused early and severe mitochondrial structural abnormalities, mitochondrial proliferation and muscle atrophy. There was dramatic reduction in mitochondrial cristae junctions and overall cristae morphology. The lack of mitochondrial cristae was accompanied by a reduction in high molecular weight mitochondrial contact site and cristae organizing system (MICOS) complexes, and to a lesser extent in OPA1. Moreover, muscles lacking ATAD3 showed altered cholesterol metabolism, accumulation of mitochondrial DNA (mtDNA) replication intermediates, progressive mtDNA depletion and deletions. Unexpectedly, decreases in the levels of some OXPHOS components occurred after cristae destabilization, indicating that ATAD3 is not crucial for mitochondrial translation, as previously suggested. Our results show a critical early role of ATAD3 in regulating mitochondrial inner membrane structure, leading to secondary defects in mtDNA replication and complex V and cholesterol levels in postmitotic tissue.This article has an associated First Person interview with the first author of the paper.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Colesterol/metabolismo , DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Músculos/metabolismo , Doenças Musculares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Replicação do DNA , DNA Mitocondrial/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Desenvolvimento Muscular , Doenças Musculares/genética , Doenças Musculares/fisiopatologiaRESUMO
Mammalian mitochondrial DNA (mtDNA) replication and repair have been studied intensively for the last 50 years. Although recently advances in elucidating the molecular mechanisms of mtDNA maintenance and the proteins involved in these have been made, there are disturbing gaps between the existing theoretical models and experimental observations. Conflicting data and hypotheses exist about the role of RNA and ribonucleotides in mtDNA replication, but also about the priming of replication and the formation of pathological rearrangements. In the presented review, we have attempted to match these loose ends and draft consensus where it can be found, while identifying outstanding issues for future research.
Assuntos
DNA Mitocondrial/genética , Mamíferos/genética , Mitocôndrias/genética , Animais , Replicação do DNA/genética , Humanos , RNA/genéticaRESUMO
Maintenance of topological homeostasis is vital for gene expression and genome replication in all organisms. Similar to other circular genomes, also mitochondrial DNA (mtDNA) is known to exist in various different topological forms, although their functional significance remains unknown. We report here that both known type II topoisomerases Top2α and Top2ß are present in mammalian mitochondria, with especially Top2ß regulating the supercoiling state of mtDNA. Loss of Top2ß or its inhibition by ciprofloxacin results in accumulation of positively supercoiled mtDNA, followed by cessation of mitochondrial transcription and replication initiation, causing depletion of mtDNA copy number. These mitochondrial effects block both cell proliferation and differentiation, possibly explaining some of the side effects associated with fluoroquinolone antibiotics. Our results show for the first time the importance of topology for maintenance of mtDNA homeostasis and provide novel insight into the mitochondrial effects of fluoroquinolones.
Assuntos
Ciprofloxacina/farmacologia , DNA Topoisomerases Tipo II/genética , DNA Mitocondrial/efeitos dos fármacos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/química , DNA Mitocondrial/genética , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas de Ligação a Poli-ADP-Ribose/química , Transcrição Gênica/efeitos dos fármacosRESUMO
Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.
Assuntos
Replicação do DNA/fisiologia , DNA Mitocondrial/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Meios de Cultura , DNA Polimerase Dirigida por DNA/genética , Fibroblastos , Deleção de Genes , Camundongos , Piridinas , Raios UltravioletaRESUMO
Like any genome, mitochondrial DNA (mtDNA) also requires the action of topoisomerases to resolve topological problems in its maintenance, but for a long time, little was known about mitochondrial topoisomerases. The last years have brought a closer insight into the function of these fascinating enzymes in mtDNA topology regulation, replication, transcription, and segregation. Here, we summarize the current knowledge about mitochondrial topoisomerases, paying special attention to mammalian mitochondrial genome maintenance. We also discuss the open gaps in the existing knowledge of mtDNA topology control and the potential involvement of mitochondrial topoisomerases in human pathologies. While Top1mt, the only exclusively mitochondrial topoisomerase in mammals, has been studied intensively for nearly a decade, only recent studies have shed some light onto the mitochondrial function of Top2ß and Top3α, enzymes that are shared between nucleus and mitochondria. Top3α mediates the segregation of freshly replicated mtDNA molecules, and its dysfunction leads to mtDNA aggregation and copy number depletion in patients. Top2ß, in contrast, regulates mitochondrial DNA replication and transcription through the alteration of mtDNA topology, a fact that should be acknowledged due to the frequent use of Topoisomerase 2 inhibitors in medical therapy.
Assuntos
DNA Topoisomerases/metabolismo , DNA Mitocondrial/metabolismo , Animais , Eucariotos/enzimologia , Humanos , Mitocôndrias/enzimologiaRESUMO
Mitochondrial DNA (mtDNA) in adult human heart is characterized by complex molecular forms held together by junctional molecules of unknown biological significance. These junctions are not present in mouse hearts and emerge in humans during postnatal development, concomitant with increased demand for oxidative metabolism. To analyze the role of mtDNA organization during oxidative stress in cardiomyocytes, we used a mouse model, which recapitulates the complex mtDNA organization of human hearts by overexpression of the mitochondrial helicase, TWINKLE. Overexpression of TWINKLE rescued the oxidative damage induced replication stalling of mtDNA, reduced mtDNA point mutation load, and modified mtDNA rearrangements in heterozygous mitochondrial superoxide dismutase knockout hearts, as well as ameliorated cardiomyopathy in mice superoxide dismutase knockout in a p21-dependent manner. We conclude that mtDNA integrity influences cell survival and reason that tissue specific modes of mtDNA maintenance represent an adaptation to oxidative stress.
Assuntos
Adaptação Biológica/fisiologia , DNA Helicases/metabolismo , DNA Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Sequência de Bases , Southwestern Blotting , Western Blotting , DNA Helicases/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA Mitocondrial/fisiologia , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/farmacologia , Dados de Sequência Molecular , Miócitos Cardíacos/fisiologia , Superóxido Dismutase/genéticaRESUMO
NOA1 is an evolutionary conserved, nuclear encoded GTPase essential for mitochondrial function and cellular survival. The function of NOA1 for assembly of mitochondrial ribosomes and regulation of OXPHOS activity depends on its GTPase activity, but so far no ligands have been identified that regulate the GTPase activity of NOA1. To identify nucleic acids that bind to the RNA-binding domain of NOA1 we employed SELEX (Systemic Evolution of Ligands by EXponential Enrichment) using recombinant mouse wildtype NOA1 and the GTPase mutant NOA1-K353R. We found that NOA1 binds specifically to oligonucleotides that fold into guanine tetrads (G-quadruplexes). Binding of G-quadruplex oligonucleotides stimulated the GTPase activity of NOA1 suggesting a regulatory link between G-quadruplex containing RNAs, NOA1 function and assembly of mitochondrial ribosomes.
Assuntos
Quadruplex G , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/enzimologia , RNA/química , RNA/metabolismo , Animais , Arginina/metabolismo , Sequência de Bases , Ativação Enzimática , GTP Fosfo-Hidrolases/isolamento & purificação , Hidrólise , Lisina/metabolismo , Camundongos , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Ligação Proteica , RNA Ribossômico/metabolismo , Proteínas Recombinantes/metabolismo , Técnica de Seleção de Aptâmeros , Especificidade por SubstratoRESUMO
Cardiomyocyte development in mammals is characterized by a transition from hyperplastic to hypertrophic growth soon after birth. The rise of cardiomyocyte cell mass in postnatal life goes along with a proportionally bigger increase in the mitochondrial mass in response to growing energy requirements. Relatively little is known about the molecular processes regulating mitochondrial biogenesis and mitochondrial DNA (mtDNA) maintenance during developmental cardiac hypertrophy. Genome-wide transcriptional profiling revealed the activation of transcriptional regulatory circuits controlling mitochondrial biogenesis in growing rat hearts. In particular, we detected a specific upregulation of factors involved in mtDNA expression and translation. More surprisingly, we found a specific upregulation of DNA repair proteins directly linked to increased oxidative damage during heart mitochondrial biogenesis, but only relatively minor changes in the mtDNA replication machinery. Our study paves the way for improved understanding of mitochondrial biogenesis, mtDNA maintenance and physiological adaptation processes in the heart and provides the first evidence for the recruitment of nucleotide excision repair proteins to mtDNA in cardiomyocytes upon DNA damage.
Assuntos
Reparo do DNA , DNA Mitocondrial/metabolismo , Coração/crescimento & desenvolvimento , Mitocôndrias Cardíacas/genética , Estresse Oxidativo , Envelhecimento/genética , Animais , Crescimento Celular , Dano ao DNA , DNA Mitocondrial/química , Regulação da Expressão Gênica , Coração/embriologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Regulação para CimaRESUMO
The non-coding regions of the mitochondrial DNAs (mtDNAs) of hares, rabbits, and pikas (Lagomorpha) contain short (â¼20 bp) and long (130-160 bp) tandem repeats, absent in related mammalian orders. In the presented study, we provide in-depth analysis for mountain hare (Lepus timidus) and brown hare (L. europaeus) mtDNA non-coding regions, together with a species- and population-level analysis of tandem repeat variation. Mountain hare short tandem repeats (SRs) as well as other analyzed hare species consist of two conserved 10 bp motifs, with only brown hares exhibiting a single, more variable motif. Long tandem repeats (LRs) also differ in sequence and copy number between species. Mountain hares have four to seven LRs, median value five, while brown hares exhibit five to nine LRs, median value six. Interestingly, introgressed mountain hare mtDNA in brown hares obtained an intermediate LR length distribution, with median copy number being the same as with conspecific brown hare mtDNA. In contrast, transfer of brown hare mtDNA into cultured mtDNA-less mountain hare cells maintained the original LR number, whereas the reciprocal transfer caused copy number instability, suggesting that cellular environment rather than the nuclear genomic background plays a role in the LR maintenance. Due to their dynamic nature and separation from other known conserved sequence elements on the non-coding region of hare mitochondrial genomes, the tandem repeat elements likely to represent signatures of ancient genetic rearrangements. clarifying the nature and dynamics of these rearrangements may shed light on the possible role of NCR repeated elements in mitochondria and in species evolution.
Assuntos
DNA Mitocondrial , Evolução Molecular , Genoma Mitocondrial , Lebres , Polimorfismo Genético , Especificidade da Espécie , Sequências de Repetição em Tandem , Animais , Lebres/genética , Sequências de Repetição em Tandem/genética , DNA Mitocondrial/genética , FilogeniaRESUMO
A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase γ (POLγA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLγA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLγA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLγA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLγA:Y955C enzyme will compete with wild-type POLγA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLγA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis.
Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação de Sentido Incorreto , Oftalmoplegia Externa Progressiva Crônica/enzimologia , Linhagem Celular , DNA Polimerase gama , Replicação do DNA , DNA Mitocondrial/genética , Humanos , Oftalmoplegia Externa Progressiva Crônica/genéticaRESUMO
The organisation of mammalian mitochondrial DNA (mtDNA) is more complex than usually assumed. Despite often being depicted as a simple circle, the topology of mtDNA can vary from supercoiled monomeric circles over catenanes and oligomers to complex multimeric networks. Replication of mtDNA is also not clear cut. Two different mechanisms of replication have been found in cultured cells and in most tissues: a strand-asynchronous mode involving temporary RNA coverage of one strand, and a strand-coupled mode rather resembling conventional nuclear DNA replication. In addition, a recombination-initiated replication mechanism is likely to be associated with the multimeric mtDNA networks found in human heart. Although an insight into the general principles and key factors of mtDNA organisation and maintenance has been gained over the last few years, there are many open questions regarding replication initiation, termination and physiological factors determining mtDNA organisation and replication mode. However, common themes in mtDNA maintenance across eukaryotic kingdoms can provide valuable lessons for future work.
Assuntos
Replicação do DNA , DNA Mitocondrial/metabolismo , Animais , DNA Catenado/metabolismo , DNA Mitocondrial/genética , Humanos , Mamíferos , Mitocôndrias Cardíacas/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Recombinação Genética , Origem de ReplicaçãoRESUMO
Two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed for nearly two decades in the analysis of replication and maintenance processes of animal mitochondrial DNA, but the method's potential has not been fully exploited. Here, we describe the various steps involved in this technique, from DNA isolation, to two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE), Southern hybridization and interpretation. We also provide examples of the applicability of 2D-AGE to investigate the different features of mtDNA maintenance and regulation.
Assuntos
Replicação do DNA , DNA Mitocondrial , Animais , DNA Mitocondrial/análise , Mitocôndrias/genética , Southern Blotting , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Ágar/métodosRESUMO
Significance: Ionizing radiation can damage cells either directly or through oxidative damage caused by ionization. Although radiation exposure from natural sources is very limited, ionizing radiation in nuclear disaster zones and long spaceflights causes inconspicuous, yet measurable physiological effects in men and animals, whose significance remains poorly known. Understanding the physiological impacts of ionizing radiation has a wide importance due to the increased use of medical imaging and radiotherapy. Recent Advances: Radiation exposure has been traditionally investigated from the perspective of DNA damage and its consequences. However, recent studies from Chernobyl as well as spaceflights have provided interesting insights into oxidative stress-induced metabolic alterations and disturbances in the circadian regulation. Critical Issues: In this review, we discuss the physiological consequences of radiation exposure in the light of oxidative stress signaling. Radiation exposure likely triggers many converging or interconnecting signaling pathways, some of which mimic mitochondrial dysfunction and might explain the observed metabolic changes. Future Directions: Better understanding of the different radiation-induced signaling pathways might help to devise strategies for mitigation of the long-term effects of radiation exposure. The utility of fibroblast growth factor 21 (FGF21) as a radiation exposure biomarker and the use of radiation hormesis as a method to protect astronauts on a prolonged spaceflight, such as a mission to Mars, should be investigated. Antioxid. Redox Signal. 37, 336-348.
Assuntos
Estresse Oxidativo , Radiação Ionizante , Animais , Dano ao DNA , Humanos , Oxirredução , Estresse Oxidativo/efeitos da radiação , Transdução de Sinais/efeitos da radiaçãoRESUMO
Bradyrhizobium denitrificans K2, isolated from an air circulation environment, has potential genes participating in inorganic nitrogen and carbon cycling. The draft genome comprises 8.31 Mb, with 7,982 coding sequences and 64.81% average G+C content. Genes related to carbon and inorganic nitrogen cycling were observed in the draft genome.