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1.
Annu Rev Biochem ; 93(1): 47-77, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38594940

RESUMEN

Mammalian mitochondrial DNA (mtDNA) is replicated and transcribed by phage-like DNA and RNA polymerases, and our understanding of these processes has progressed substantially over the last several decades. Molecular mechanisms have been elucidated by biochemistry and structural biology and essential in vivo roles established by cell biology and mouse genetics. Single molecules of mtDNA are packaged by mitochondrial transcription factor A into mitochondrial nucleoids, and their level of compaction influences the initiation of both replication and transcription. Mutations affecting the molecular machineries replicating and transcribing mtDNA are important causes of human mitochondrial disease, reflecting the critical role of the genome in oxidative phosphorylation system biogenesis. Mechanisms controlling mtDNA replication and transcription still need to be clarified, and future research in this area is likely to open novel therapeutic possibilities for treating mitochondrial dysfunction.


Asunto(s)
Replicación del ADN , ADN Mitocondrial , Transcripción Genética , Humanos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Animales , Mitocondrias/metabolismo , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Mutación , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética
2.
Nat Rev Mol Cell Biol ; 25(2): 119-132, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37783784

RESUMEN

The expression of mitochondrial genes is regulated in response to the metabolic needs of different cell types, but the basic mechanisms underlying this process are still poorly understood. In this Review, we describe how different layers of regulation cooperate to fine tune initiation of both mitochondrial DNA (mtDNA) transcription and replication in human cells. We discuss our current understanding of the molecular mechanisms that drive and regulate transcription initiation from mtDNA promoters, and how the packaging of mtDNA into nucleoids can control the number of mtDNA molecules available for both transcription and replication. Indeed, a unique aspect of the mitochondrial transcription machinery is that it is coupled to mtDNA replication, such that mitochondrial RNA polymerase is additionally required for primer synthesis at mtDNA origins of replication. We discuss how the choice between replication-primer formation and genome-length RNA synthesis is controlled at the main origin of replication (OriH) and how the recent discovery of an additional mitochondrial promoter (LSP2) in humans may change this long-standing model.


Asunto(s)
Replicación del ADN , Transcripción Genética , Humanos , Replicación del ADN/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo
3.
EMBO Mol Med ; 15(5): e16775, 2023 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-37013609

RESUMEN

Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.


Asunto(s)
Enfermedades Mitocondriales , Enfermedades Musculares , Humanos , Mitocondrias/genética , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Síndrome , Inestabilidad Genómica
4.
Mol Cell ; 82(19): 3646-3660.e9, 2022 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-36044900

RESUMEN

The human mitochondrial genome must be replicated and expressed in a timely manner to maintain energy metabolism and supply cells with adequate levels of adenosine triphosphate. Central to this process is the idea that replication primers and gene products both arise via transcription from a single light strand promoter (LSP) such that primer formation can influence gene expression, with no consensus as to how this is regulated. Here, we report the discovery of a second light strand promoter (LSP2) in humans, with features characteristic of a bona fide mitochondrial promoter. We propose that the position of LSP2 on the mitochondrial genome allows replication and gene expression to be orchestrated from two distinct sites, which expands our long-held understanding of mitochondrial gene expression in humans.


Asunto(s)
Genoma Mitocondrial , Adenosina Trifosfato/metabolismo , ADN Mitocondrial/metabolismo , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Transcripción Genética
5.
Cell ; 185(13): 2309-2323.e24, 2022 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-35662414

RESUMEN

The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.


Asunto(s)
ADN Mitocondrial , Proteínas Mitocondriales , Animales , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Dimerización , Humanos , Mamíferos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN/metabolismo , ARN Mitocondrial , ARN Citoplasmático Pequeño , Partícula de Reconocimiento de Señal , Transcripción Genética
6.
Nat Commun ; 13(1): 3701, 2022 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-35764627

RESUMEN

Stress granules (SGs) are non-membranous organelles facilitating stress responses and linking the pathology of age-related diseases. In a genome-wide imaging-based phenomic screen, we identify Pab1 co-localizing proteins under 2-deoxy-D-glucose (2-DG) induced stress in Saccharomyces cerevisiae. We find that deletion of one of the Pab1 co-localizing proteins, Lsm7, leads to a significant decrease in SG formation. Under 2-DG stress, Lsm7 rapidly forms foci that assist in SG formation. The Lsm7 foci form via liquid-liquid phase separation, and the intrinsically disordered region and the hydrophobic clusters within the Lsm7 sequence are the internal driving forces in promoting Lsm7 phase separation. The dynamic Lsm7 phase-separated condensates appear to work as seeding scaffolds, promoting Pab1 demixing and subsequent SG initiation, seemingly mediated by RNA interactions. The SG initiation mechanism, via Lsm7 phase separation, identified in this work provides valuable clues for understanding the mechanisms underlying SG formation and SG-associated human diseases.


Asunto(s)
Fenómenos Bioquímicos , Proteínas de Saccharomyces cerevisiae , Gránulos Citoplasmáticos/metabolismo , Humanos , Proteínas de Unión a Poli(A)/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Gránulos de Estrés
7.
Nucleic Acids Res ; 50(2): 989-999, 2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-35018464

RESUMEN

Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.


Asunto(s)
ADN Polimerasa gamma/metabolismo , ADN/metabolismo , Mitocondrias/genética , ARN Mitocondrial/metabolismo , Ribonucleótidos/metabolismo , Replicación del ADN , Escherichia coli/genética , Humanos
8.
Sci Adv ; 7(27)2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34215584

RESUMEN

We report a role for the mitochondrial single-stranded DNA binding protein (mtSSB) in regulating mitochondrial DNA (mtDNA) replication initiation in mammalian mitochondria. Transcription from the light-strand promoter (LSP) is required both for gene expression and for generating the RNA primers needed for initiation of mtDNA synthesis. In the absence of mtSSB, transcription from LSP is strongly up-regulated, but no replication primers are formed. Using deep sequencing in a mouse knockout model and biochemical reconstitution experiments with pure proteins, we find that mtSSB is necessary to restrict transcription initiation to optimize RNA primer formation at both origins of mtDNA replication. Last, we show that human pathological versions of mtSSB causing severe mitochondrial disease cannot efficiently support primer formation and initiation of mtDNA replication.


Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , Animales , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Mamíferos/genética , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo
10.
Nat Commun ; 12(1): 1135, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33602924

RESUMEN

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Mitocondrias/genética , Mutación/genética , Enfermedades del Sistema Nervioso/genética , Transcripción Genética , Adolescente , Adulto , Niño , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/química , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Lactante , Masculino , Enfermedades del Sistema Nervioso/patología , Fosforilación Oxidativa , Linaje , Dominios Proteicos , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adulto Joven
11.
PLoS Genet ; 16(12): e1009242, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33315859

RESUMEN

Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.


Asunto(s)
ADN Mitocondrial/genética , Eliminación de Gen , Duplicación de Gen , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Animales , ADN Mitocondrial/química , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Ratones , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/normas
12.
Nature ; 588(7839): 712-716, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33328633

RESUMEN

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.


Asunto(s)
Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Microscopía por Crioelectrón , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Genes Mitocondriales/efectos de los fármacos , Humanos , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Especificidad por Sustrato/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Crit Rev Biochem Mol Biol ; 55(6): 509-524, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32972254

RESUMEN

Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome (mtDNA) that codes for subunits of the oxidative phosphorylation machinery. Mutations in mtDNA cause a number of rare, human disorders and are also associated with more common conditions, such as neurodegeneration and biological aging. In this review, we discuss our current understanding of mtDNA replication in mammalian cells and how this process is regulated. We also discuss how deletions can be formed during mtDNA replication.


Asunto(s)
ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN Mitocondrial/genética , Humanos , Proteínas Mitocondriales/genética
14.
PLoS Genet ; 16(5): e1008798, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32469861

RESUMEN

Alterations in epigenetic silencing have been associated with ageing and tumour formation. Although substantial efforts have been made towards understanding the mechanisms of gene silencing, novel regulators in this process remain to be identified. To systematically search for components governing epigenetic silencing, we developed a genome-wide silencing screen for yeast (Saccharomyces cerevisiae) silent mating type locus HMR. Unexpectedly, the screen identified the mismatch repair (MMR) components Pms1, Mlh1, and Msh2 as being required for silencing at this locus. We further found that the identified genes were also required for proper silencing in telomeres. More intriguingly, the MMR mutants caused a redistribution of Sir2 deacetylase, from silent mating type loci and telomeres to rDNA regions. As a consequence, acetylation levels at histone positions H3K14, H3K56, and H4K16 were increased at silent mating type loci and telomeres but were decreased in rDNA regions. Moreover, knockdown of MMR components in human HEK293T cells increased subtelomeric DUX4 gene expression. Our work reveals that MMR components are required for stable inheritance of gene silencing patterns and establishes a link between the MMR machinery and the control of epigenetic silencing.


Asunto(s)
Homólogo 1 de la Proteína MutL/genética , Proteínas MutL/genética , Proteína 2 Homóloga a MutS/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilación , Reparación de la Incompatibilidad de ADN , Epigénesis Genética , Silenciador del Gen , Genes del Tipo Sexual de los Hongos , Herencia , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Telómero/genética
15.
Proc Natl Acad Sci U S A ; 117(13): 7524-7535, 2020 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-32184324

RESUMEN

Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.


Asunto(s)
Respiración de la Célula/fisiología , Fermentación/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Etanol/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Glucosa/metabolismo , Espectrometría de Masas/métodos , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Mol Cell ; 76(5): 784-796.e6, 2019 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-31588022

RESUMEN

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.


Asunto(s)
Proteínas 14-3-3/metabolismo , Biomarcadores de Tumor/metabolismo , Exorribonucleasas/metabolismo , Mitocondrias/enzimología , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Mitocondrial/metabolismo , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Animales , Biomarcadores de Tumor/genética , Exorribonucleasas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mitocondrial/genética , Células Sf9 , Spodoptera
18.
EMBO Rep ; 20(6)2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31036713

RESUMEN

Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.


Asunto(s)
Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Procesamiento Postranscripcional del ARN , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo , Animales , ADN Mitocondrial , Desarrollo Embrionario/genética , Eliminación de Gen , Regulación de la Expresión Génica , Sitios Genéticos , Heterocigoto , Ratones , Ratones Noqueados , Mitocondrias/ultraestructura , Fenotipo , Regiones Promotoras Genéticas
19.
Nat Commun ; 10(1): 759, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30770810

RESUMEN

Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.


Asunto(s)
ADN Mitocondrial/genética , Eliminación de Secuencia/genética , Southern Blotting , Replicación del ADN/genética , Humanos , Proteínas Mitocondriales/genética , Mutación/genética
20.
PLoS Genet ; 15(1): e1007781, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30605451

RESUMEN

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3'-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.


Asunto(s)
Replicación del ADN/genética , ADN Mitocondrial/biosíntesis , Mitocondrias/genética , Ribonucleasa H/genética , Animales , ADN Polimerasa gamma/genética , ADN Mitocondrial/genética , Proteínas de Unión al ADN/genética , Humanos , Ratones , Origen de Réplica/genética
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