Howard T Jacobs

University of Helsinki, Helsinki, Uusimaa, Finland

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Publications (131)940.26 Total impact

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    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 07/2015; DOI:10.1073/pnas.1503653112 · 9.81 Impact Factor
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    ABSTRACT: Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elon-gation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.
    PLoS Genetics 02/2015; · 8.52 Impact Factor
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    ABSTRACT: Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.
    PLoS Genetics 02/2015; 11(2):e1004985. DOI:10.1371/journal.pgen.1004985 · 8.17 Impact Factor
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    ABSTRACT: Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous enzymes that catalyze the reversible hydration reaction of carbon dioxide. CAs are present as six structurally divergent enzyme families: α, β, γ, δ, ζ and η. β-CAs have a wide distribution across different species including invertebrates. Previously, we showed that Drosophila melanogaster β-CA is a highly active mitochondrial enzyme. In this study, we investigated the function of Drosophila β-CA by silencing the expression of the β-CA gene using UAS/GAL4-based RNA interference (RNAi) in Drosophila in vivo. Crossing β-CA RNAi lines over ubiquitous Actin driver flies did not produce any viable progeny, indicating that β-CA expression is required for fly development. RNAi silencing of β-CA ubiquitously in adult flies did not affect their survival rate or function of mitochondrial electron transport chain. Importantly, β-CA RNAi led to impaired reproduction. All β-CA knockdown females were sterile, and produced few or no eggs. Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization. These results indicate that although Drosophila β-CA is dispensable for survival of adult flies, it is essential for female fertility.
    Frontiers in Zoology 01/2015; 12:19. DOI:10.1186/s12983-015-0111-3 · 2.30 Impact Factor
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    ABSTRACT: An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was only able to weakly complement the phenotypes produced by the knockdown of complex I subunits.
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 11/2014; 1837(11). DOI:10.1016/j.bbabio.2014.08.001 · 4.83 Impact Factor
  • Ian J. Holt · Howard T. Jacobs
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    ABSTRACT: Last year, we reported a new mechanism of DNA replication in mammals. It occurs inside mitochondria and entails the use of processed transcripts, termed bootlaces, which hybridize with the displaced parental strand as the replication fork advances. Here we discuss possible reasons why such an unusual mechanism of DNA replication might have evolved. The bootlace mechanism can minimize the occurrence and impact of single-strand breaks that would otherwise threaten genome stability. Furthermore, by providing an implicit mismatch recognition system, it should limit the occurrence of replication-dependent deletions and insertions, and defend against invading elements. Such a mechanism may also limit attempts to manipulate the mammalian mitochondrial genome.
    BioEssays 09/2014; 36(11). DOI:10.1002/bies.201400052 · 4.84 Impact Factor
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    Kia K Kemppainen · Esko Kemppainen · Howard T Jacobs
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    ABSTRACT: A point mutation (technical knockout(25t), tko(25t)) in the Drosophila gene coding for mitoribosomal protein S12 generates a phenotype of developmental delay and bang-sensitivity. tko(25t) has been intensively studied as an animal model for human mitochondrial diseases associated with deficiency of mitochondrial protein synthesis and consequent multiple respiratory chain defects. Transgenic expression in Drosophila of the alternative oxidase, AOX, derived from Ciona intestinalis, has previously been shown to mitigate the toxicity of respiratory chain inhibitors, and to rescue mutant and knockdown phenotypes associated with cytochrome oxidase deficiency. We therefore tested whether AOX expression could compensate the mutant phenotype of tko(25t), using the GeneSwitch system to activate expression at different times in development. The developmental delay of tko(25t) was not mitigated by expression of AOX throughout development. AOX expression for one day following eclosion, or continuously throughout development, had no effect on the bang-sensitivity of tko(25t) adults, and continued expression in adults over 30 days also produced no amelioration of the phenotype. In contrast, transgenic expression of the yeast alternative NADH dehydrogenase Ndi1 was synthetically semi-lethal with tko(25t), and lethal when combined with both AOX and tko(25t). We conclude that AOX does not rescue tko(25t) and that the mutant phenotype is not due solely to limitations on electron flow in the respiratory chain, rather to a more complex metabolic defect. The future therapeutic use of AOX in disorders of mitochondrial translation may thus be of limited value.
    G3-Genes Genomes Genetics 08/2014; 4(10). DOI:10.1534/g3.114.013946 · 2.51 Impact Factor
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e71-e72. DOI:10.1016/j.bbabio.2014.05.013 · 4.83 Impact Factor
  • Howard T. Jacobs
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e57-e58. DOI:10.1016/j.bbabio.2014.05.226 · 4.83 Impact Factor
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    ABSTRACT: The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number.
    Molecular Systems Biology 06/2014; 10(6). DOI:10.15252/msb.20145117 · 14.10 Impact Factor
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    ABSTRACT: A point mutation (stress-sensitive B(1), sesB(1) in the Drosophila gene coding for the major adult isoform of the adenine nuclear translocase (ANT) represents a model for human diseases associated with ANT insufficiency. We characterized the organismal, bioenergetic and molecular phenotype of sesB(1), then tested strategies to compensate the mutant phenotype. In addition to developmental delay and bang-sensitivity, sesB(1) manifests impaired response to sound, defective male courtship, female sterility and curtailed lifespan. These phenotypes, apart from the last two, are shared with the tko(25t) mutant in mitoribosomal protein S12. Mitochondria from sesB(1) adults showed a decreased respiratory control ratio and downregulation of cytochrome oxidase. sesB(1) adults exhibited ATP depletion, lactate accumulation, and changes in gene expression consistent with a metabolic shift towards glycolysis, with activation of lactate dehydrogenase and anaplerotic pathways. Females also showed downregulation of many genes required for oogenesis, and their eggs, though fertilized, failed to develop to the larval stages. The sesB(1) phenotypes of developmental delay and bang-sensitivity were alleviated by altered mtDNA background. Female sterility was substantially rescued by somatic expression of the alternative oxidase (AOX) from Ciona intestinalis, whereas AOX did not alleviate developmental delay. Our findings illustrate the potential of different therapeutic strategies for ANT-linked diseases, based on increasing mitochondrial bioenergy production, or on alleviating metabolic stress.
    Disease Models and Mechanisms 05/2014; 7(6). DOI:10.1242/dmm.016527 · 5.54 Impact Factor
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    ABSTRACT: Mitochondrial disorders are nowadays recognized as impinging on most areas of medicine. They include specific and widespread organ involvement, including both tissue degeneration or tumor formation. Despite the spectacular progresses made in the identification of their underlying molecular basis, effective therapy remains a distant goal. Our still rudimentary understanding of the patholophysiological mechanisms by which these diseases arise constitutes an obstacle to developing any rational treatments. In this context, the idea of using a heterologous gene, encoding a supplemental oxidase otherwise absent from mammals, potentially by-passing the defective portion of the respiratory chain, has been proposed more than 10 years ago. The recent progress made in the expression of the alternative oxidase (AOX) in a wide range of biological systems and disease conditions reveals great potential benefit, considering the broad impact of mitochondrial diseases. This review addresses the state-of-the-art and the perspectives that can be now envisioned by using this strategy.
    British Journal of Pharmacology 01/2014; 171(8). DOI:10.1111/bph.12570 · 4.99 Impact Factor
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    ABSTRACT: Mitochondrial dysfunction is a significant factor in human disease, ranging from systemic disorders of childhood to cardiomyopathy, ischemia and neurodegeneration. Cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, is a frequent target. Lower eukaryotes possess alternative respiratory-chain enzymes which provide non proton-translocating bypasses for respiratory complexes I (single-subunit NADH dehydrogenases, e.g. Ndi1 from yeast) or III+IV (alternative oxidase, AOX), under conditions of respiratory stress or overload. In previous studies it was shown that transfer of yeast Ndi1 or Ciona intestinalis AOX to Drosophila was able to overcome the lethality produced by toxins or partial knockdown of complexes I or IV. Here we show that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different cIV subunits, including integral subunits required for catalysis, as well as peripheral subunits required for multimerization and assembly. AOX was also able to overcome the pupal lethality produced by muscle-specific knockdown of subunit CoVb, although the rescued flies were short-lived and had a motility defect. cIV knockdown in neurons was not lethal during development but produced a rapidly progressing locomotor and seizure-sensitivity phenotype which was substantially alleviated by AOX. Expression of Ndi1 exacerbated the neuronal phenotype produced by cIV knockdown. Ndi1 expressed in place of essential cI subunits, produced a distinct residual phenotype of delayed development, bang-sensitivity and male sterility. These findings confirm the potential utility of alternative respiratory chain enzymes as tools to combat mitochondrial disease, whilst indicating important limitations thereof.
    Human Molecular Genetics 11/2013; 23(8). DOI:10.1093/hmg/ddt601 · 6.68 Impact Factor
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    ABSTRACT: All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.
    PLoS Genetics 09/2013; 9(9):e1003800. DOI:10.1371/journal.pgen.1003800 · 8.17 Impact Factor
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    ABSTRACT: 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.
    Nucleic Acids Research 04/2013; 41(7). DOI:10.1093/nar/gkt196 · 9.11 Impact Factor
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    ABSTRACT: Proliferating cells require coordinated gene expression between the nucleus and mitochondria in order to divide, ensuring sufficient organelle number in daughter cells [1]. However, the machinery and mechanisms whereby proliferating cells monitor mitochondria and coordinate organelle biosynthesis remain poorly understood. Antibiotics inhibiting mitochondrial translation have emerged as therapeutics for human cancers because they block cell proliferation [2, 3]. These proliferative defects were attributable to modest decreases in mitochondrial respiration [3, 4], even though tumors are mainly glycolytic [5] and mitochondrial respiratory chain function appears to play a minor role in cell proliferation in vivo [6]. Here we challenge this interpretation by demonstrating that one class of antiproliferative antibiotic induces stalled mitochondrial ribosomes, which triggers a mitochondrial ribosome and RNA decay pathway. Rescue of the stalled mitochondrial ribosomes initiates a retrograde signaling response to block cell proliferation and occurs prior to any loss of mitochondrial respiration. The loss of respiratory chain function is simply a downstream effect of impaired mitochondrial translation and not the antiproliferative signal. This mitochondrial ribosome quality-control pathway is actively monitored in cells and constitutes an important organelle checkpoint for cell division.
    Current biology: CB 02/2013; 23(6). DOI:10.1016/j.cub.2013.02.019 · 9.92 Impact Factor
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    Priit Jõers · Howard T Jacobs
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    ABSTRACT: Mitochondrial DNA synthesis is necessary for the normal function of the organelle and for the eukaryotic organism as a whole. Here we demonstrate, using two-dimensional agarose gel electrophoresis to analyse replication intermediates, that unidirectional, strand-coupled DNA synthesis is the prevalent mode of mtDNA replication in Drosophila melanogaster. Commencing within the single, extended non-coding region (NCR), replication proceeds around the circular genome, manifesting an irregular rate of elongation, and pausing frequently in specific regions. Evidence for a limited contribution of strand-asynchronous DNA synthesis was found in a subset of mtDNA molecules, but confined to the ribosomal RNA gene region, just downstream of the NCR. Our findings imply that strand-coupled replication is widespread amongst metazoans, and should inform future research on mtDNA metabolism in D. melanogaster.
    PLoS ONE 01/2013; 8(1):e53249. DOI:10.1371/journal.pone.0053249 · 3.23 Impact Factor
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    ABSTRACT: Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.
    PLoS Genetics 01/2013; 9(1):e1003182. DOI:10.1371/journal.pgen.1003182 · 8.17 Impact Factor
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    ABSTRACT: Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an “emergency shutdown” system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.
    Journal of Biological Chemistry 11/2012; 287(46). DOI:10.1074/jbc.M112.386060 · 4.57 Impact Factor
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Publication Stats

6k Citations
940.26 Total Impact Points

Institutions

  • 2013–2015
    • University of Helsinki
      Helsinki, Uusimaa, Finland
  • 1999–2014
    • Tampere University Hospital (TAUH)
      Tammerfors, Province of Western Finland, Finland
  • 1997–2013
    • University of Tampere
      • • Institute of Biomedical Technology
      • • Institute of Medical Technology
      Tammerfors, Province of Western Finland, Finland
  • 1987–2009
    • University of Glasgow
      • Division of Genetics
      Glasgow, Scotland, United Kingdom
  • 2004
    • Roswell Park Cancer Institute
      Buffalo, New York, United States
  • 2000
    • University of Dundee
      Dundee, Scotland, United Kingdom