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Justin C. St. John - One of the best experts on this subject based on the ideXlab platform.
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the control of Mitochondrial DNA Replication during development and tumorigenesis
2015Co-Authors: Justin C. St. John, William LeeAbstract:Mitochondrial DNA (mtDNA) copy number is strictly regulated during development and tumorigenesis. Pluripotent stem cells and cancer stem-like cells use glycolysis for energy metabolism, as they possess low mtDNA copy number, which promotes cell proliferation. As pluripotent stem cells can differentiate into all cell types of the body, they establish the mtDNA set point during early development, maintaining mtDNA copy number at low levels but enabling differentiating cells to acquire the appropriate numbers of mtDNA copy to meet their specific demands for OXPHOS-derived ATP, as they become specialized cells. This process is mediated by changes to DNA methylation at exon 2 of the catalytic subunit of the Mitochondrial-specific polymerase, POLGA. Cancer stem-like cells, however, are hypermethylated and maintain low mtDNA copy number, resulting in their dependence on aerobic glycolysis. Their hypermethylation at exon 2 of POLGA also promotes their multipotent state. As a result, cancer cells are unable to increase their mtDNA content and differentiate into specific lineages unless they are treated with DNA demethylation agents or partially depleted of their mtDNA. This review describes these processes in depth and argues that DNA methylation of POLGA is instrumental in the fate of pluripotent stem cells and cancer cells.
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The Effects of Nuclear Reprogramming on Mitochondrial DNA Replication
2013Co-Authors: Richard D. W. Kelly, Huseyin Sumer, Matthew Mckenzie, Joao Facucho-oliveira, Ian A. Trounce, Paul J. Verma, Justin C. St. JohnAbstract:Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of Mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA Replication factors, which initiate a major Replication event on Day 6 of differentiation. Here, we examined mtDNA Replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific Replication factors, PolgA and Peo1 , were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA Replication during pluripotency but this can be re-established through the use of epigenetic modifiers.
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Mitochondrial DNA Replication during differentiation of murine embryonic stem cells
2007Co-Authors: Joao M Facuchooliveira, Emma C Spikings, Jon Alderson, Stuart Egginton, Justin C. St. JohnAbstract:1. 1. Facucho-Oliveira J. M., 2. et al. 2007. J. Cell Sci. doi:10.1242/jcs.016972 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft.jtitle%253DJ.%2BCell%2BSci.%26rft_id%253Dinfo%253Adoi%252F10.1242%252Fjcs.016972%26rft_id%253Dinfo%253Apmid%252F17971411%
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Mitochondrial DNA Replication during differentiation of murine embryonic stem cells.
2007Co-Authors: Joao M Facucho-oliveira, Jon Alderson, Emma C Spikings, Stuart Egginton, Justin C. St. JohnAbstract:Oxidative phosphorylation (OXPHOS), the intracellular process that generates the majority of the ATP of a cell through the electron-transfer chain, is highly dependent on proteins encoded by the Mitochondrial genome (mtDNA). MtDNA Replication is regulated by the nuclear-encoded Mitochondrial transcription factor A (TFAM) and the Mitochondrial-specific DNA polymerase gamma, which consists of a catalytic (POLG) and an accessory (POLG2) subunit. Differentiation of pluripotent embryonic stem cells (ESCs) into specific cell types requires expansion of discrete populations of mitochondria and mtDNA Replication to meet the specific metabolic requirements of the cell. We determined by real-time PCR that expression of pluripotent markers is reduced before the upregulation of Polg, Polg2 and Tfam in spontaneously differentiating R1 murine (m)ESCs, along with transient increases in mtDNA copy number. In D3 mESCs, the initial transient increase did not take place. However, precursors of neuronal and cardiomyocyte differentiation were positive for both POLG and TFAM. Similar-stage ESCs also showed active mtDNA Replication, identified by 5-bromo-2'-deoxy-uridine labelling, as mtDNA copy number increased. Retinoic-acid-induced differentiation resulted in more consistent patterns of Replication and upregulation of Polg, Polg2 and Tfam, whereas siRNA knockdown demonstrated that steady-state expression of POLG is essential for maintaining pluripotency.
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regulated Mitochondrial DNA Replication during oocyte maturation is essential for successful porcine embryonic development
2007Co-Authors: Emma C Spikings, Jon Alderson, Justin C. St. JohnAbstract:Cellular ATP is mainly generated through Mitochondrial oxidative phosphorylation, which is dependent on Mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded Mitochondrial Replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, Mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA Replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA Replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA Replication during oocyte maturation is essential for successful embryonic development.
Maria Falkenberg - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial DNA Replication in mammalian cells overview of the pathway
2018Co-Authors: Maria FalkenbergAbstract:Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome and a dedicated DNA Replication machinery is required for its maintenance. Many disease-causing mutations affect Mitochondrial Replication factors and a detailed understanding of the Replication process may help to explain the pathogenic mechanisms underlying a number of Mitochondrial diseases. We here give a brief overview of DNA Replication in mammalian mitochondria, describing our current understanding of this process and some unanswered questions remaining.
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primer removal during mammalian Mitochondrial DNA Replication
2015Co-Authors: Jay P Uhler, Maria FalkenbergAbstract:The small circular Mitochondrial genome in mammalian cells is replicated by a dedicated replisome, defects in which can cause Mitochondrial disease in humans. A fundamental step in Mitochondrial DNA (mtDNA) Replication and maintenance is the removal of the RNA primers needed for Replication initiation. The nucleases RNase H1, FEN1, DNA2, and MGME1 have been implicated in this process. Here we review the role of these nucleases in the light of primer removal pathways in mitochondria, highlight associations with disease, as well as consider the implications for mtDNA Replication initiation.
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Regulation of DNA Replication at the end of the Mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element
2015Co-Authors: Elisabeth Jemt, Örjan Persson, Marcela Dávila López, Christoph Freyer, Jay P Uhler, Majda Mehmedovic, Tore Samuelsson, Claes M Gustafsson, Maria FalkenbergAbstract:The majority of Mitochondrial DNA Replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the Mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5′-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3′-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the Replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and Replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length Mitochondrial DNA Replication.
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the exonuclease activity of DNA polymerase γ is required for ligation during Mitochondrial DNA Replication
2015Co-Authors: Bertil Macao, Jay P Uhler, Claes M Gustafsson, James B Stewart, Triinu Siibak, Xuefeng Zhu, Yonghong Shi, Wenwen Sheng, Monica Olsson, Maria FalkenbergAbstract:Mitochondrial DNA (mtDNA) polymerase γ (POLγ) harbours a 3'-5' exonuclease proofreading activity. Here we demonstrate that this activity is required for the creation of ligatable ends during mtDNA Replication. Exonuclease-deficient POLγ fails to pause on reaching a downstream 5'-end. Instead, the enzyme continues to polymerize into double-stranded DNA, creating an unligatable 5'-flap. Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation. In mice, inactivation of the exonuclease activity causes an increase in mtDNA mutations and premature ageing phenotypes. These mutator mice also contain high levels of truncated, linear fragments of mtDNA. We demonstrate that the formation of these fragments is due to impaired ligation, causing nicks near the origin of heavy-strand DNA Replication. In the subsequent round of Replication, the nicks lead to double-strand breaks and linear fragment formation.
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in vivo occupancy of Mitochondrial single stranded DNA binding protein supports the strand displacement mode of DNA Replication
2014Co-Authors: Javier Miralles Fuste, Elisabeth Jemt, Örjan Persson, Claes M Gustafsson, Sjoerd Wanrooij, Xuefeng Zhu, Yonghong Shi, Nasim Sabouri, Maria FalkenbergAbstract:Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA Replication. We here demonstrate that the Mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA Replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA Replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the Mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the Replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for Mitochondrial DNA Replication.
William C Copeland - One of the best experts on this subject based on the ideXlab platform.
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role of the Mitochondrial DNA Replication machinery in Mitochondrial DNA mutagenesis aging and age related diseases
2017Co-Authors: Karen L Debalsi, Kirsten E Hoff, William C CopelandAbstract:As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA Replication machinery. Here, we review evidence that errors caused by this Replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in Mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that Mitochondrial dysfunction, in conjunction with altered Mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve Mitochondrial function and attenuate the symptoms of aging are examined.
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Human Mitochondrial DNA Replication machinery and disease.
2016Co-Authors: Matthew J. Young, William C CopelandAbstract:The human Mitochondrial genome is replicated by DNA polymerase γ in concert with key components of the Mitochondrial DNA (mtDNA) Replication machinery. Defects in mtDNA Replication or nucleotide metabolism cause deletions, point mutations, or depletion of mtDNA. The resulting loss of cellular respiration ultimately induces Mitochondrial genetic diseases, including mtDNA depletion syndromes (MDS) such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders such as progressive external ophthalmoplegia, ataxia-neuropathy, or Mitochondrial neurogastrointestinal encephalomyopathy. Here we review the current literature regarding human mtDNA Replication and heritable disorders caused by genetic changes of the POLG, POLG2, Twinkle, RNASEH1, DNA2, and MGME1 genes.
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Defects of Mitochondrial DNA Replication
2014Co-Authors: William C CopelandAbstract:Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the Mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in Mitochondrial DNA Replication or nucleotide metabolism can cause Mitochondrial genetic diseases due to Mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include Mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and Mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or Mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of Mitochondrial DNA Replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of Mitochondrial DNA and Mitochondrial disease.
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Defects in Mitochondrial DNA Replication and human disease
2011Co-Authors: William C CopelandAbstract:Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase g in concert with accessory proteins such as the mtDNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA Replication arise from the Mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA Replication or nucleotide metabolism can cause Mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA Replication (POLG, POLG2, C10orf2) and nucleotide metabol...
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Mitochondrial DNA Replication and disease: Insights from DNA polymerase γ mutations
2011Co-Authors: Jeffrey D. Stumpf, William C CopelandAbstract:DNA polymerase γ (pol γ), encoded by POLG, is responsible for replicating human Mitochondrial DNA. About 150 mutations in the human POLG have been identified in patients with Mitochondrial diseases such as Alpers syndrome, progressive external ophthalmoplegia, and ataxia-neuropathy syndromes. Because many of the mutations are described in single citations with no genotypic family history, it is important to ascertain which mutations cause or contribute to Mitochondrial disease. The vast majority of data about POLG mutations has been generated from biochemical characterizations of recombinant pol γ. However, recently, the study of Mitochondrial dysfunction in Saccharomyces cerevisiae and mouse models provides important in vivo evidence for the role of POLG mutations in disease. Also, the published 3D-structure of the human pol γ assists in explaining some of the biochemical and genetic properties of the mutants. This review summarizes the current evidence that identifies and explains disease-causing POLG mutations.
Takehiro Yasukawa - One of the best experts on this subject based on the ideXlab platform.
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an overview of mammalian Mitochondrial DNA Replication mechanisms
2018Co-Authors: Takehiro Yasukawa, Dongchon KangAbstract:While the majority of DNA is enclosed within the nucleus, the mitochondria also contain their own, separate DNA, the Mitochondrial DNA (mtDNA). Mutations in mtDNA are associated with various human diseases, demonstrating the importance of mtDNA. Intensive studies over the last 18 years have demonstrated the presence of two distinct classes of mtDNA Replication intermediates in mammals. One involves leading-strand DNA synthesis in the absence of synchronous lagging-strand DNA synthesis. Currently there are competing models in which the lagging-strand template is either systematically hybridized to processed Mitochondrial transcripts, or coated with protein, until the lagging-strand DNA synthesis takes place. The other class of mtDNA Replication intermediates has many properties of conventional, coupled leading- and lagging-strand DNA synthesis. Additionally, the highly unusual arrangement of DNA in human heart mitochondria suggests a third mechanism of Replication. These findings indicate that the mtDNA Replication systems of humans and other mammals are far more complex than previously thought, and thereby will require further research to understand the full picture of mtDNA Replication.
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pathological ribonuclease h1 causes r loop depletion and aberrant DNA segregation in mitochondria
2016Co-Authors: Gokhan Akman, Takehiro Yasukawa, Laura J. Bailey, Bradley J Holmes, Chloe F Moss, Radha Desai, Ilaria Dalla Rosa, Romina Durigon, Mara MennuniAbstract:The genetic information in mammalian Mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its Replication and expression. Many molecules of Mitochondrial DNA bear a third strand of DNA, known as “7S DNA,” which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the Mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for Mitochondrial DNA Replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with Mitochondrial disease, R-loops are of low abundance, and there is Mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of Mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.
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Mitochondrial DNA Replication proceeds via a bootlace mechanism involving the incorporation of processed transcripts
2013Co-Authors: Aurelio Reyes, Lawrence Kazak, Takehiro Yasukawa, Howard T Jacobs, Stuart R. Wood, Ian J HoltAbstract:The observation that long tracts of RNA are associated with replicating molecules of Mitochondrial DNA (mtDNA) suggests that the Mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing Replication intermediates are chased into mature mtDNA, to which they are thus in precursor–product relationship. While a DNA chain terminator rapidly blocks the labeling of Mitochondrial Replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA Replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA Replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA Replication, and that it involves a ‘bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the Replication fork advances.
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involvement of DNA ligase iii and ribonuclease h1 in Mitochondrial DNA Replication in cultured human cells
2011Co-Authors: Heini Ruhanen, Kathy Ushakov, Takehiro YasukawaAbstract:Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian Mitochondrial DNA (mtDNA) Replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and Replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA Replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the Mitochondrial Replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.
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mammalian Mitochondrial DNA Replication intermediates are essentially duplex but contain extensive tracts of rna DNA hybrid
2010Co-Authors: Jaakko L. O. Pohjoismäki, Aurelio Reyes, Takehiro Yasukawa, Mingyao Yang, Stuart R. Wood, Laura J. Bailey, Tricia J. Cluett, Steffi Goffart, Bradley J Holmes, Smaranda WillcoxAbstract:We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact Mitochondrial DNA Replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of Mitochondrial DNA Replication involving RNA incorporation throughout the lagging strand.
Howard T Jacobs - One of the best experts on this subject based on the ideXlab platform.
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developmental arrest in drosophila melanogaster caused by Mitochondrial DNA Replication defects cannot be rescued by the alternative oxidase
2018Co-Authors: Ana Paula Rodrigues, Howard T Jacobs, Andre Ferreira De Camargo, Ana Andjelkovic, Marcos T OliveiraAbstract:The xenotopic expression of the alternative oxidase AOX from the tunicate Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of Mitochondrial respiratory chain defects. AOX is a non-proton pumping, Mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of Mitochondrial DNA (mtDNA) Replication defects caused by disturbances in the mtDNA helicase or DNA polymerase γ. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko25t, a fly mutant with Mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of Mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human Mitochondrial diseases.
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primer retention owing to the absence of rnase h1 is catastrophic for Mitochondrial DNA Replication
2015Co-Authors: Howard T Jacobs, Stuart R. Wood, Bradley J Holmes, Gokhan Akman, Kiran Sakhuja, Susana M Cerritelli, Chloe F Moss, Mark BowmakerAbstract:Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for Mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in Mitochondrial DNA Replication. Analysis of replicating Mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in Mitochondrial DNA. However, the retained primers present an obstacle to the Mitochondrial DNA polymerase γ in subsequent rounds of Replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in Mitochondrial DNA Replication is the removal of primers at the origin of Replication.
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Mitochondrial DNA Replication proceeds via a bootlace mechanism involving the incorporation of processed transcripts
2013Co-Authors: Aurelio Reyes, Lawrence Kazak, Takehiro Yasukawa, Howard T Jacobs, Stuart R. Wood, Ian J HoltAbstract:The observation that long tracts of RNA are associated with replicating molecules of Mitochondrial DNA (mtDNA) suggests that the Mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing Replication intermediates are chased into mature mtDNA, to which they are thus in precursor–product relationship. While a DNA chain terminator rapidly blocks the labeling of Mitochondrial Replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA Replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA Replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA Replication, and that it involves a ‘bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the Replication fork advances.
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overexpression of mterfd1 or mterfd3 impairs the completion of Mitochondrial DNA Replication
2011Co-Authors: Anne K Hyvarinen, Jaakko L. O. Pohjoismäki, Ian J Holt, Howard T JacobsAbstract:The physiological roles of the Mitochondrial transcription termination factor (mTERF) family are poorly understood. MTERF and its homologues influence transcriptional readthrough in vitro, but the extent to which they regulate Mitochondrial RNA levels in vivo is unclear. In addition, MTERF was previously shown to promote Replication pausing. To test their roles in mtDNA metabolism, we created cell-lines inducibly expressing epitope-tagged versions of two members of the mTERF family, MTERFD1 and MTERFD3, as well as shRNA constructs targeted at each. We confirmed Mitochondrial targeting and lack of sequence-specific DNA binding for both factors. Over-expression of epitope-tagged MTERFD1 or MTERFD3 resulted in modest mtDNA copy-number depletion and an accumulation of specific mtDNA Replication intermediates indicating an impairment of the terminal steps of Replication. These findings further implicate the mTERF family in restraining Replication fork progression and support the idea that they facilitate the orderly passage of Replication and transcription machineries, thus contributing to genome stability.
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a bidirectional origin of Replication maps to the major noncoding region of human Mitochondrial DNA
2005Co-Authors: Takehiro Yasukawa, Mingyao Yang, Howard T Jacobs, Ian J HoltAbstract:Summary In solid tissues of vertebrates, initiation of Mitochondrial DNA Replication encompasses a broad zone downstream of the major noncoding region (NCR). In contrast, analysis with two-dimensional agarose gel electrophoresis of Mitochondrial DNA Replication intermediates in cultured human cells revealed initiation concentrated in the NCR. Mapping of prominent free 5′ ends on the heavy strand of Mitochondrial DNA identified two clusters of potential start sites. One mapped to the previously assigned origin of strand-asynchronous Replication (O H ); the other lay several hundred nucleotides away from O H , toward the other end of the NCR. The latter cluster is proposed to be the major site of bidirectional Replication initiation on the basis of the following: its prominence is enhanced in cells amplifying Mitochondrial DNA after experimentally induced Mitochondrial DNA depletion; free 5′ ends are found in corresponding positions on the opposite strand; it is transient in nature; and it is associated with bubble arcs.