Publications by authors named "Johannes N Spelbrink"

51 Publications

RNA Crosslinking to Analyze the Mitochondrial RNA-Binding Proteome.

Methods Mol Biol 2021 ;2192:147-158

Radboud Center for Mitochondrial Medicine, Department of Paediatrics, Radboudumc, Nijmegen, The Netherlands.

Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.
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http://dx.doi.org/10.1007/978-1-0716-0834-0_12DOI Listing
March 2021

A Combined Mass Spectrometry and Data Integration Approach to Predict the Mitochondrial Poly(A) RNA Interacting Proteome.

Front Cell Dev Biol 2019 15;7:283. Epub 2019 Nov 15.

Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Centre, Nijmegen, Netherlands.

In order to synthesize the 13 oxidative phosphorylation proteins encoded by mammalian mtDNA, a large assortment of nuclear encoded proteins is required. These include mitoribosomal proteins and various RNA processing, modification and degradation enzymes. RNA crosslinking has been successfully applied to identify whole-cell poly(A) RNA-binding proteomes, but this method has not been adapted to identify mitochondrial poly(A) RNA-binding proteomes. Here we developed and compared two related methods that specifically enrich for mitochondrial poly(A) RNA-binding proteins and analyzed bound proteins using mass spectrometry. To obtain a catalog of the mitochondrial poly(A) RNA interacting proteome, we used Bayesian data integration to combine these two mitochondrial-enriched datasets as well as published whole-cell datasets of RNA-binding proteins with various online resources, such as mitochondrial localization from MitoCarta 2.0 and co-expression analyses. Our integrated analyses ranked the complete human proteome for the likelihood of mtRNA interaction. We show that at a specific, inclusive cut-off of the corrected false discovery rate (cFDR) of 69%, we improve the number of predicted proteins from 185 to 211 with our mass spectrometry data as input for the prediction instead of the published whole-cell datasets. The chosen cut-off determines the cFDR: the less proteins included, the lower the cFDR will be. For the top 100 proteins, inclusion of our data instead of the published whole-cell datasets improve the cFDR from 54% to 31%. We show that the mass spectrometry method most specific for mitochondrial RNA-binding proteins involves 4-thiouridine labeling followed by mitochondrial isolation with subsequent UV-crosslinking.
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http://dx.doi.org/10.3389/fcell.2019.00283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873792PMC
November 2019

Dominant mutations in mtDNA maintenance gene SSBP1 cause optic atrophy and foveopathy.

J Clin Invest 2020 01;130(1):143-156

Institute of Neurosciences of Montpellier, INSERM, University of Montpellier, Montpellier, France.

Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDSs), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in single-strand binding protein 1 (SSBP1) in 5 unrelated families, leading to the R38Q and R107Q amino acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a revised SSBP1 crystal structure. Structural analysis suggested that both mutations affect dimer interactions and presumably distort the DNA-binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication, and induces mtDNA depletion. Our study showing that mutations in SSBP1 cause a form of dominant optic atrophy frequently accompanied with foveopathy brings insights into mtDNA maintenance disorders.
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http://dx.doi.org/10.1172/JCI128513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934222PMC
January 2020

Mitochondrial RNA granules are critically dependent on mtDNA replication factors Twinkle and mtSSB.

Nucleic Acids Res 2019 04;47(7):3680-3698

Radboud Center for Mitochondrial Medicine, Department of Paediatrics, Radboudumc, Nijmegen, The Netherlands.

Newly synthesized mitochondrial RNA is concentrated in structures juxtaposed to nucleoids, called RNA granules, that have been implicated in mitochondrial RNA processing and ribosome biogenesis. Here we show that two classical mtDNA replication factors, the mtDNA helicase Twinkle and single-stranded DNA-binding protein mtSSB, contribute to RNA metabolism in mitochondria and to RNA granule biology. Twinkle colocalizes with both mitochondrial RNA granules and nucleoids, and it can serve as bait to greatly enrich established RNA granule proteins, such as G-rich sequence factor 1, GRSF1. Likewise, mtSSB also is not restricted to the nucleoids, and repression of either mtSSB or Twinkle alters mtRNA metabolism. Short-term Twinkle depletion greatly diminishes RNA granules but does not inhibit RNA synthesis or processing. Either mtSSB or GRSF1 depletion results in RNA processing defects, accumulation of mtRNA breakdown products as well as increased levels of dsRNA and RNA:DNA hybrids. In particular, the processing and degradation defects become more pronounced with both proteins depleted. These findings suggest that Twinkle is essential for RNA organization in granules, and that mtSSB is involved in the recently proposed GRSF1-mtRNA degradosome pathway, a route suggested to be particularly aimed at degradation of G-quadruplex prone long non-coding mtRNAs.
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http://dx.doi.org/10.1093/nar/gkz047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468249PMC
April 2019

Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication.

Nucleic Acids Res 2018 11;46(20):10771-10781

Department of Clinical Movement Neurosciences, Institute of Neurology, Royal Free Campus, University College London, London NW3 2PF, UK.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle.
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http://dx.doi.org/10.1093/nar/gky852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237803PMC
November 2018

The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair.

Sci Rep 2018 03 29;8(1):5368. Epub 2018 Mar 29.

Radboud Center for Mitochondrial Medicine, Department of Paediatrics, Radboudumc, Nijmegen, The Netherlands.

EXD2 is a recently identified exonuclease that has been implicated in nuclear double-strand break repair. Given our long standing interest in mitochondrial DNA maintenance and indications that EXD2 could also be a mitochondrial protein we sought to determine its cellular localization and possible mitochondrial associated functions. Our results show that EXD2 indeed shows mitochondrial localization, but, surprisingly, is found predominantly associated with the mitochondrial outer-membrane. Gradient purified nuclei show only the faintest hint of EXD2 presence while overexpression of the predicted full-length protein shows exclusive mitochondrial localization. Importantly, induction of double-strand DNA breaks via X-irradiation or Zeocin treatment does not support the notion that EXD2 re-locates to the nucleus following double-strand breaks and thus is unlikely to have a direct role in nuclear DNA repair. Knockdown or overexpression of EXD2 affects the cellular distribution of mitochondria. These results suggest that the reported defects in nuclear DNA repair following EXD2 depletion are likely an indirect consequence of altered mitochondrial dynamics and/or function.
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http://dx.doi.org/10.1038/s41598-018-23690-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876329PMC
March 2018

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Brain 2017 Jun;140(6):1595-1610

Department of Pediatrics, Saitama Medical University, Moroyama-machi, Iruma-gun, Saitama, Japan.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.
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http://dx.doi.org/10.1093/brain/awx094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445257PMC
June 2017

A homozygous missense mutation in ERAL1, encoding a mitochondrial rRNA chaperone, causes Perrault syndrome.

Hum Mol Genet 2017 07;26(13):2541-2550

Department of Clinical Genetics, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands.

Perrault syndrome (PS) is a rare recessive disorder characterized by ovarian dysgenesis and sensorineural deafness. It is clinically and genetically heterogeneous, and previously mutations have been described in different genes, mostly related to mitochondrial proteostasis. We diagnosed three unrelated females with PS and set out to identify the underlying genetic cause using exome sequencing. We excluded mutations in the known PS genes, but identified a single homozygous mutation in the ERAL1 gene (c.707A > T; p.Asn236Ile). Since ERAL1 protein binds to the mitochondrial 12S rRNA and is involved in the assembly of the small mitochondrial ribosomal subunit, the identified variant represented a likely candidate. In silico analysis of a 3D model for ERAL1 suggested that the mutated residue hinders protein-substrate interactions, potentially affecting its function. On a molecular basis, PS skin fibroblasts had reduced ERAL1 protein levels. Complexome profiling of the cells showed an overall decrease in the levels of assembled small ribosomal subunit, indicating that the ERAL1 variant affects mitochondrial ribosome assembly. Moreover, levels of the 12S rRNA were reduced in the patients, and were rescued by lentiviral expression of wild type ERAL1. At the physiological level, mitochondrial respiration was markedly decreased in PS fibroblasts, confirming disturbed mitochondrial function. Finally, knockdown of the C. elegans ERAL1 homologue E02H1.2 almost completely blocked egg production in worms, mimicking the compromised fertility in PS-affected women. Our cross-species data in patient cells and worms support the hypothesis that mutations in ERAL1 can cause PS and are associated with changes in mitochondrial metabolism.
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http://dx.doi.org/10.1093/hmg/ddx152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965403PMC
July 2017

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

Mol Cell 2016 Jan 24;61(2):199-209. Epub 2015 Dec 24.

Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.
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http://dx.doi.org/10.1016/j.molcel.2015.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724312PMC
January 2016

Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure.

Sci Rep 2015 Oct 19;5:15292. Epub 2015 Oct 19.

Nijmegen Centre for Mitochondrial Disorders, RadboudUMC, Nijmegen, The Netherlands.

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.
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http://dx.doi.org/10.1038/srep15292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609938PMC
October 2015

The hexameric structure of the human mitochondrial replicative helicase Twinkle.

Nucleic Acids Res 2015 Apr 30;43(8):4284-95. Epub 2015 Mar 30.

Structural MitoLab; Department of Structural Biology, Molecular Biology Institute Barcelona (IBMB-CSIC), Barcelona, E-08028, Spain

The mitochondrial replicative helicase Twinkle is involved in strand separation at the replication fork of mitochondrial DNA (mtDNA). Twinkle malfunction is associated with rare diseases that include late onset mitochondrial myopathies, neuromuscular disorders and fatal infantile mtDNA depletion syndrome. We examined its 3D structure by electron microscopy (EM) and small angle X-ray scattering (SAXS) and built the corresponding atomic models, which gave insight into the first molecular architecture of a full-length SF4 helicase that includes an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD) and a RecA-like hexamerization C-terminal domain (CTD). The EM model of Twinkle reveals a hexameric two-layered ring comprising the ZBDs and RPDs in one layer and the CTDs in another. In the hexamer, contacts in trans with adjacent subunits occur between ZBDs and RPDs, and between RPDs and CTDs. The ZBDs show important structural heterogeneity. In solution, the scattering data are compatible with a mixture of extended hexa- and heptameric models in variable conformations. Overall, our structural data show a complex network of dynamic interactions that reconciles with the structural flexibility required for helicase activity.
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http://dx.doi.org/10.1093/nar/gkv189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417153PMC
April 2015

The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

Sci Rep 2015 Mar 17;5:9149. Epub 2015 Mar 17.

Institute of Internal Medicine and Gastroenterology, Catholic University of the Sacred Heart School of Medicine.

Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5-5 μM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.
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http://dx.doi.org/10.1038/srep09149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361992PMC
March 2015

Whole cell formaldehyde cross-linking simplifies purification of mitochondrial nucleoids and associated proteins involved in mitochondrial gene expression.

PLoS One 2015 19;10(2):e0116726. Epub 2015 Feb 19.

Mitochondrial DNA Maintenance Group, BioMediTech, FI-33014 University of Tampere, Tampere, Finland; Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Geert Grooteplein 10, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116726PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335056PMC
January 2016

CLPB mutations cause 3-methylglutaconic aciduria, progressive brain atrophy, intellectual disability, congenital neutropenia, cataracts, movement disorder.

Am J Hum Genet 2015 Feb 15;96(2):245-57. Epub 2015 Jan 15.

Institute of Neurogenetics, University of Lübeck, 23562 Lübeck, Germany.

We studied a group of individuals with elevated urinary excretion of 3-methylglutaconic acid, neutropenia that can develop into leukemia, a neurological phenotype ranging from nonprogressive intellectual disability to a prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, and early death. Exome sequencing of two unrelated individuals and subsequent Sanger sequencing of 16 individuals with an overlapping phenotype identified a total of 14 rare, predicted deleterious alleles in CLPB in 14 individuals from 9 unrelated families. CLPB encodes caseinolytic peptidase B homolog ClpB, a member of the AAA+ protein family. To evaluate the relevance of CLPB in the pathogenesis of this syndrome, we developed a zebrafish model and an in vitro assay to measure ATPase activity. Suppression of clpb in zebrafish embryos induced a central nervous system phenotype that was consistent with cerebellar and cerebral atrophy that could be rescued by wild-type, but not mutant, human CLPB mRNA. Consistent with these data, the loss-of-function effect of one of the identified variants (c.1222A>G [p.Arg408Gly]) was supported further by in vitro evidence with the mutant peptides abolishing ATPase function. Additionally, we show that CLPB interacts biochemically with ATP2A2, known to be involved in apoptotic processes in severe congenital neutropenia (SCN) 3 (Kostmann disease [caused by HAX1 mutations]). Taken together, mutations in CLPB define a syndrome with intellectual disability, congenital neutropenia, progressive brain atrophy, movement disorder, cataracts, and 3-methylglutaconic aciduria.
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http://dx.doi.org/10.1016/j.ajhg.2014.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320260PMC
February 2015

DNA sequences proximal to human mitochondrial DNA deletion breakpoints prevalent in human disease form G-quadruplexes, a class of DNA structures inefficiently unwound by the mitochondrial replicative Twinkle helicase.

J Biol Chem 2014 Oct 5;289(43):29975-93. Epub 2014 Sep 5.

From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, NIH Biomedical Research Center, Baltimore, Maryland 21224,

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the "Pattern Finder" G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.
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http://dx.doi.org/10.1074/jbc.M114.567073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208006PMC
October 2014

Quality matters: how does mitochondrial network dynamics and quality control impact on mtDNA integrity?

Philos Trans R Soc Lond B Biol Sci 2014 Jul;369(1646):20130442

Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Geert Grooteplein 10, PO Box 9101, 6500 HB Nijmegen, The Netherlands FinMIT Centre of Excellence, Institute of Biomedical Technology and Tampere University Hospital, Pirkanmaa Hospital District, 33014 Tampere, Finland

Mammalian mtDNA encodes for 13 core proteins of oxidative phosphorylation. Mitochondrial DNA mutations and deletions cause severe myopathies and neuromuscular diseases. Thus, the integrity of mtDNA is pivotal for cell survival and health of the organism. We here discuss the possible impact of mitochondrial fusion and fission on mtDNA maintenance as well as positive and negative selection processes. Our focus is centred on the important question of how the quality of mtDNA nucleoids can be assured when selection and mitochondrial quality control works on functional and physiological phenotypes constituted by oxidative phosphorylation proteins. The organelle control theory suggests a link between phenotype and nucleoid genotype. This is discussed in the light of new results presented here showing that mitochondrial transcription factor A/nucleoids are restricted in their intramitochondrial mobility and probably have a limited sphere of influence. Together with recent published work on mitochondrial and mtDNA heteroplasmy dynamics, these data suggest first, that single mitochondria might well be internally heterogeneous and second, that nucleoid genotypes might be linked to local phenotypes (although the link might often be leaky). We discuss how random or site-specific mitochondrial fission can isolate dysfunctional parts and enable their elimination by mitophagy, stressing the importance of fission in the process of mtDNA quality control. The role of fusion is more multifaceted and less understood in this context, but the mixing and equilibration of matrix content might be one of its important functions.
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http://dx.doi.org/10.1098/rstb.2013.0442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032518PMC
July 2014

What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations.

Philos Trans R Soc Lond B Biol Sci 2014 Jul;369(1646):20130438

Behaviour and Genetics of Social Insects Lab, The University of Sydney, Sydney NSW 2006, Australia.

The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is relaxed. Furthermore, because mtDNA replication is not strictly regulated, within-cell selection may favour mtDNA variants with a replication advantage, but a deleterious effect on cell fitness. The opportunities for selfish mtDNA mutations to spread are restricted by various organism-level adaptations, such as uniparental transmission, germline mtDNA bottlenecks, germline selection and, during somatic growth, regular alternation between fusion and fission of mitochondria. These mechanisms are all hypothesized to maintain functional mtDNA. However, the strength of selection for maintenance of functional mtDNA progressively declines with age, resulting in age-related diseases. Furthermore, organismal adaptations that most probably evolved to restrict the opportunities for selfish mtDNA create secondary problems. Owing to predominantly maternal mtDNA transmission, recombination among mtDNA from different individuals is highly restricted or absent, reducing the scope for repair. Moreover, maternal inheritance precludes selection against mtDNA variants with male-specific effects. We finish by discussing the consequences of life-history differences among taxa with respect to mtDNA evolution and make a case for the use of microorganisms to experimentally manipulate levels of selection.
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http://dx.doi.org/10.1098/rstb.2013.0438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032515PMC
July 2014

Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication.

Nucleic Acids Res 2014 Jan 25;42(2):952-67. Epub 2013 Oct 25.

FinMIT Centre of Excellence, Institute of Biomedical Technology & Tampere University Hospital, Pirkanmaa Hospital District, FI-33014 University of Tampere, Finland and Department of Pediatrics, Institute for Genetic and Metabolic Disease, Radboud University Medical Centre, Geert Grooteplein 10, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

Mitochondrial DNA (mtDNA) is organized in discrete protein-DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.
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http://dx.doi.org/10.1093/nar/gkt988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3902951PMC
January 2014

To be or not to be a nucleoid protein: a comparison of mass-spectrometry based approaches in the identification of potential mtDNA-nucleoid associated proteins.

Biochimie 2014 May 25;100:219-26. Epub 2013 Sep 25.

Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands; Institute of Biomedical Technology & Tampere University Hospital, Pirkanmaa Hospital District, University of Tampere, FI-33014, Finland. Electronic address:

In the last decade it has become increasingly clear that mitochondrial DNA (mtDNA) is not naked but associated with proteins in poorly defined structures called nucleoids that are essential for mtDNA maintenance. The function of nucleoids is not simply to package mtDNA but also to provide a stable environment for its replication, transcription and repair. Even though their properties and dynamics have begun to be revealed in recent years, their structural and molecular organization remains largely unknown in mammals. Although, there are a number of proteins identified to be nucleoid associated by using several biochemical isolation methods combined with mass spectrometric analysis, the main difficulties in the identification of these proteins are their low abundance and the assumed dynamic composition of nucleoids. Considering various purification methods, there is a thin line between the stringency and specificity in the identification of potential nucleoid associated proteins. In this review, the main focus is to provide a comprehensive comparison of the so far published purification and analysis methods to generate a list of potentially nucleoid associated proteins, but also, to discuss the disadvantages and possible improvements in proteomic analyses.
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http://dx.doi.org/10.1016/j.biochi.2013.09.017DOI Listing
May 2014

Analysis of 953 human proteins from a mitochondrial HEK293 fraction by complexome profiling.

PLoS One 2013 23;8(7):e68340. Epub 2013 Jul 23.

Department of Laboratory Medicine, Laboratory of Genetic Endocrine and Metabolic Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands.

Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification-mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068340PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720734PMC
March 2014

Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness.

Nat Genet 2012 Jun 10;44(7):797-802. Epub 2012 Jun 10.

Department of Pediatrics, Radboud University Nijmegen Medical Centre (RUNMC), Nijmegen, The Netherlands.

Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking.
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http://dx.doi.org/10.1038/ng.2325DOI Listing
June 2012

Mytoe: automatic analysis of mitochondrial dynamics.

Bioinformatics 2012 Apr 9;28(7):1050-1. Epub 2012 Feb 9.

Department of Signal Processing, Tampere University of Technology, FI-33520 Tampere, Finland.

Summary: We present Mytoe, a tool for analyzing mitochondrial morphology and dynamics from fluorescence microscope images. The tool provides automated quantitative analysis of mitochondrial motion by optical flow estimation and of morphology by segmentation of individual branches of the network-like structure of the organelles. Mytoe quantifies several features of individual branches, such as length, tortuosity and speed, and of the macroscopic structure, such as mitochondrial area and degree of clustering. We validate the methods and apply them to the analysis of sequences of images of U2OS human cells with fluorescently labeled mitochondria.

Availability: Source code, Windows software and Manual available at http://www.cs.tut.fi/%7Esanchesr/mito

Supplementary Information: Supplementary data are available at Bioinformatics online.

Contact: [email protected]; [email protected]
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http://dx.doi.org/10.1093/bioinformatics/bts073DOI Listing
April 2012

Replication stalling by catalytically impaired Twinkle induces mitochondrial DNA rearrangements in cultured cells.

Mitochondrion 2011 Jul 20;11(4):630-4. Epub 2011 Apr 20.

Department of Cardiac Development and Remodelling, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany.

Pathological mitochondrial DNA (mtDNA) rearrangements have been proposed to result from repair of double-strand breaks caused by blockage of mitochondrial DNA (mtDNA) replication. As mtDNA deletions are seen only in post-mitotic tissues, it has been suggested that they are selected out in actively dividing cells. By electron microscopy we observed rearranged mtDNA molecules in cultured human cells expressing a catalytically impaired helicase. As these molecules were undetectable by PCR, we propose that deleted mtDNA molecules in cultured cells are fragile and sensitive to heating. Further consequences of mtDNA replication stalling are discussed.
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http://dx.doi.org/10.1016/j.mito.2011.04.002DOI Listing
July 2011

Sequence-specific stalling of DNA polymerase γ and the effects of mutations causing progressive ophthalmoplegia.

Hum Mol Genet 2011 Mar 12;20(6):1212-23. Epub 2011 Jan 12.

Division of Metabolic Diseases, Karolinska Institutet, Solna, SE-17177 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1093/hmg/ddq565DOI Listing
March 2011

Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid.

J Mol Biol 2010 Apr 23;397(5):1144-55. Epub 2010 Feb 23.

Department of Forensic Medicine and Tampere University Hospital, FI-33014 University of Tampere, Finland.

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.
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http://dx.doi.org/10.1016/j.jmb.2010.02.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857715PMC
April 2010

Functional organization of mammalian mitochondrial DNA in nucleoids: history, recent developments, and future challenges.

IUBMB Life 2010 Jan;62(1):19-32

FinMIT Centre of Excellence, Institute of Medical Technology and Tampere University Hospital, University of Tampere, Tampere 33014 TAY, Finland.

Various proteins involved in replication, repair, and the structural organization of mitochondrial DNA (mtDNA) have been characterized in detail over the past 25 or so years. In addition, in recent years, many proteins were identified with a role in the dynamics of the mitochondrial network. Using advanced imaging and an increasing number of cytological techniques, we have begun to realize that an important aspect to mtDNA maintenance, in both health and disease, is its organization within the dynamic mitochondrial network in discrete protein-DNA complexes usually termed nucleoids. Here, I review recent developments in the study of nucleoid dynamics and proteins. I will discuss the implications of the organization of mtDNA in nucleoids in light of DNA replication, repair, gene expression, segregation, and inheritance.
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http://dx.doi.org/10.1002/iub.282DOI Listing
January 2010

Human Dna2 is a nuclear and mitochondrial DNA maintenance protein.

Mol Cell Biol 2009 Aug 1;29(15):4274-82. Epub 2009 Jun 1.

Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.
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http://dx.doi.org/10.1128/MCB.01834-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715806PMC
August 2009

A new splice variant of the mouse SIRT3 gene encodes the mitochondrial precursor protein.

PLoS One 2009 31;4(3):e4986. Epub 2009 Mar 31.

University of Tampere, Institute of Medical Technology and Tampere University Hospital, Tampere, Finland.

Background: Mammals have seven NAD-dependent protein deacetylases. These proteins, called sirtuins, are homologous to yeast Sir2, and are emerging as important regulators of lifespan and intermediary metabolism. Three mammalian sirtuins, SIRT3-5 are mitochondrial. Sirtuins are highly conserved between species, yet mouse SIRT3 was reported to be markedly shorter than its human counterpart and to lack the N-terminal mitochondrial targeting signal present in the human protein.

Results: We have isolated a novel mouse SIRT3 splice variant. This cDNA contains two translation initiation codons upstream of the originally reported start site. We show, using immunofluorescence and protein expression analysis that these longer variants are expressed and efficiently targeted to mitochondria, and that the processed forms of these longer variants are identical in size to the endogenous mouse SIRT3. We also show that the previously described form of SIRT3 is not mitochondrial.

Conclusions: Our observations point to a high level of conservation of SIRT3 as a mitochondrial protein in mice and human and indicate that several previous studies, which addressed mouse Sirt3 function, need to be re-evaluated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004986PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659428PMC
May 2009

Twinkle mutations associated with autosomal dominant progressive external ophthalmoplegia lead to impaired helicase function and in vivo mtDNA replication stalling.

Hum Mol Genet 2009 Jan 29;18(2):328-40. Epub 2008 Oct 29.

Institute of Medical Technology and Tampere University Hospital, Biokatu 6, 33014, Tampere, Finland.

Mutations in the mitochondrial helicase Twinkle underlie autosomal dominant progressive external ophthalmoplegia (PEO), as well as recessively inherited infantile-onset spinocerebellar ataxia and rare forms of mitochondrial DNA (mtDNA) depletion syndrome. Familial PEO is typically associated with the occurrence of multiple mtDNA deletions, but the mechanism by which Twinkle dysfunction induces deletion formation has been under debate. Here we looked at the effects of Twinkle adPEO mutations in human cell culture and studied the mtDNA replication in the Deletor mouse model, which expresses a dominant PEO mutation in Twinkle and accumulates multiple mtDNA deletions during life. We show that expression of dominant Twinkle mutations results in the accumulation of mtDNA replication intermediates in cell culture. This indicated severe replication pausing or stalling and caused mtDNA depletion. A strongly enhanced accumulation of replication intermediates was evident also in six-week-old Deletor mice compared with wild-type littermates, even though mtDNA deletions accumulate in a late-onset fashion in this model. In addition, our results in cell culture pointed to a problem of transcription that preceded the mtDNA depletion phenotype and might be of relevance in adPEO pathophysiology. Finally, in vitro assays showed functional defects in the various Twinkle mutants and broadly agreed with the cell culture phenotypes such as the level of mtDNA depletion and the level of accumulation of replication intermediates. On the basis of our results we suggest that mtDNA replication pausing or stalling is the common consequence of Twinkle PEO mutations that predisposes to multiple deletion formation.
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http://dx.doi.org/10.1093/hmg/ddn359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2638771PMC
January 2009
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