Publications by authors named "William C Copeland"

100 Publications

Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase.

Bio Protoc 2021 Sep 5;11(17):e4139. Epub 2021 Sep 5.

Physics Department, North Carolina State University, Raleigh, North Carolina, USA.

Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.
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http://dx.doi.org/10.21769/BioProtoc.4139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8443461PMC
September 2021

Mechanisms of SSBP1 variants in mitochondrial disease: Molecular dynamics simulations reveal stable tetramers with altered DNA binding surfaces.

DNA Repair (Amst) 2021 Nov 17;107:103212. Epub 2021 Aug 17.

Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 27709, USA. Electronic address:

Several mutations in the gene for the mitochondrial single stranded DNA binding protein (SSBP1) have recently been implicated in human disease, but initial reports are insufficient to explain the molecular mechanism of disease, including the possible role of SSBP1 heterotetramers in heterozygous patients. Here we employed molecular simulations to model the dynamics of wild type and 31 variant SSBP1 tetramer systems, including 7 variant homotetramer and 24 representative heterotetramer systems. Our simulations indicate that all variants are stable and most have stronger intermonomer interactions, reduced solvent accessible surface areas, and a net loss of positive surface charge. We then used structural alignments and phosphate binding simulations to predict DNA binding surfaces on SSBP1. Our models suggest that nearly the entire surface of SSBP1, excluding flexible loops and protruding helices, is available for DNA binding, and we observed several potential DNA binding hotspots. Changes to the protein surface in variant SSBP1 tetramers potentially alter anchor points or wrapping paths, rather than abolishing binding altogether. Overall, our findings disqualify tetramer destabilization or gross disruption of DNA binding as mechanisms of disease. Instead, they are consistent with subtle changes to DNA binding, wrapping, or release that cause rare but consequential failures of mtDNA maintenance, which, in turn, are consistent with the late onset of disease in most of the reported SSBP1 cases.
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http://dx.doi.org/10.1016/j.dnarep.2021.103212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8526412PMC
November 2021

Polymerase γ efficiently replicates through many natural template barriers but stalls at the HSP1 quadruplex.

J Biol Chem 2020 12;295(51):17802-17815

Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, USA. Electronic address:

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex-forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.
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http://dx.doi.org/10.1074/jbc.RA120.015390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762954PMC
December 2020

Consequences of compromised mitochondrial genome integrity.

DNA Repair (Amst) 2020 09;93:102916

Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, NC, 27709, USA. Electronic address:

Maintenance and replication of the mitochondrial genome (mtDNA) is essential to mitochondrial function and eukaryotic energy production through the electron transport chain. mtDNA is replicated by a core set of proteins: Pol γ, Twinkle, and the single-stranded DNA binding protein. Fewer pathways exist for repair of mtDNA than nuclear DNA, and unrepaired damage to mtDNA may accumulate and lead to dysfunctional mitochondria. The mitochondrial genome is susceptible to damage by both endogenous and exogenous sources. Missense mutations to the nuclear genes encoding the core mtDNA replisome (POLG, POLG2, TWNK, and SSBP1) cause changes to the biochemical functions of their protein products. These protein variants can damage mtDNA and perturb oxidative phosphorylation. Ultimately, these mutations cause a diverse set of diseases that can affect virtually every system in the body. Here, we briefly review the mechanisms of mtDNA damage and the clinical consequences of disease variants of the core mtDNA replisome.
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http://dx.doi.org/10.1016/j.dnarep.2020.102916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587307PMC
September 2020

Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging.

Genome Biol 2020 09 17;21(1):248. Epub 2020 Sep 17.

Genome Integrity and Structural Biology Laboratory, Mitochondrial DNA Replication Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA.

Background: Acquired human mitochondrial genome (mtDNA) deletions are symptoms and drivers of focal mitochondrial respiratory deficiency, a pathological hallmark of aging and late-onset mitochondrial disease.

Results: To decipher connections between these processes, we create LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. LostArc reveals 35 million deletions (~ 470,000 unique spans) in skeletal muscle from 22 individuals with and 19 individuals without pathogenic variants in POLG. This nuclear gene encodes the catalytic subunit of replicative mitochondrial DNA polymerase γ. Ablation, the deleted mtDNA fraction, suffices to explain skeletal muscle phenotypes of aging and POLG-derived disease. Unsupervised bioinformatic analyses reveal distinct age- and disease-correlated deletion patterns.

Conclusions: These patterns implicate replication by DNA polymerase γ as the deletion driver and suggest little purifying selection against mtDNA deletions by mitophagy in postmitotic muscle fibers. Observed deletion patterns are best modeled as mtDNA deletions initiated by replication fork stalling during strand displacement mtDNA synthesis.
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http://dx.doi.org/10.1186/s13059-020-02138-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500033PMC
September 2020

Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase.

J Biol Chem 2020 04 25;295(17):5564-5576. Epub 2020 Mar 25.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709. Electronic address:

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA-binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.
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http://dx.doi.org/10.1074/jbc.RA120.012795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186178PMC
April 2020

SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder.

J Clin Invest 2020 01;130(1):108-125

Pathology Unit, Department of Laboratories, Bambino Gesù Children's Hospital, Rome, Italy.

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.
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http://dx.doi.org/10.1172/JCI128514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934201PMC
January 2020

Mitochondrial single-stranded DNA binding protein novel de novo SSBP1 mutation in a child with single large-scale mtDNA deletion (SLSMD) clinically manifesting as Pearson, Kearns-Sayre, and Leigh syndromes.

PLoS One 2019 3;14(9):e0221829. Epub 2019 Sep 3.

Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, United States of America.

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband's fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1. Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719858PMC
March 2020

POLG-related disorders and their neurological manifestations.

Nat Rev Neurol 2019 01;15(1):40-52

Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Durham, NC, USA.

The POLG gene encodes the mitochondrial DNA polymerase that is responsible for replication of the mitochondrial genome. Mutations in POLG can cause early childhood mitochondrial DNA (mtDNA) depletion syndromes or later-onset syndromes arising from mtDNA deletions. POLG mutations are the most common cause of inherited mitochondrial disorders, with as many as 2% of the population carrying these mutations. POLG-related disorders comprise a continuum of overlapping phenotypes with onset from infancy to late adulthood. The six leading disorders caused by POLG mutations are Alpers-Huttenlocher syndrome, which is one of the most severe phenotypes; childhood myocerebrohepatopathy spectrum, which presents within the first 3 years of life; myoclonic epilepsy myopathy sensory ataxia; ataxia neuropathy spectrum; autosomal recessive progressive external ophthalmoplegia; and autosomal dominant progressive external ophthalmoplegia. This Review describes the clinical features, pathophysiology, natural history and treatment of POLG-related disorders, focusing particularly on the neurological manifestations of these conditions.
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http://dx.doi.org/10.1038/s41582-018-0101-0DOI Listing
January 2019

Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein.

Nucleic Acids Res 2018 11;46(21):11287-11302

Genome Integrity and Structural Biology Laboratory, NIEHS, NIH, Research Triangle Park, NC 27709, USA.

Improper maintenance of the mitochondrial genome progressively disrupts cellular respiration and causes severe metabolic disorders commonly termed mitochondrial diseases. Mitochondrial single-stranded DNA binding protein (mtSSB) is an essential component of the mtDNA replication machinery. We utilized single-molecule methods to examine the modes by which human mtSSB binds DNA to help define protein interactions at the mtDNA replication fork. Direct visualization of individual mtSSB molecules by atomic force microscopy (AFM) revealed a random distribution of mtSSB tetramers bound to extended regions of single-stranded DNA (ssDNA), strongly suggesting non-cooperative binding by mtSSB. Selective binding to ssDNA was confirmed by AFM imaging of individual mtSSB tetramers bound to gapped plasmid DNA substrates bearing defined single-stranded regions. Shortening of the contour length of gapped DNA upon binding mtSSB was attributed to DNA wrapping around mtSSB. Tracing the DNA path in mtSSB-ssDNA complexes with Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy established a predominant binding mode with one DNA strand winding once around each mtSSB tetramer at physiological salt conditions. Single-molecule imaging suggests mtSSB may not saturate or fully protect single-stranded replication intermediates during mtDNA synthesis, leaving the mitochondrial genome vulnerable to chemical mutagenesis, deletions driven by primer relocation or other actions consistent with clinically observed deletion biases.
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http://dx.doi.org/10.1093/nar/gky875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265486PMC
November 2018

Characterization of the human homozygous R182W POLG2 mutation in mitochondrial DNA depletion syndrome.

PLoS One 2018 29;13(8):e0203198. Epub 2018 Aug 29.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC, United States of America.

Mutations in mitochondrial DNA (mtDNA) have been linked to a variety of metabolic, neurological and muscular diseases which can present at any time throughout life. MtDNA is replicated by DNA polymerase gamma (Pol γ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB). The Pol γ holoenzyme is a heterotrimer consisting of the p140 catalytic subunit and a p55 homodimeric accessory subunit encoded by the nuclear genes POLG and POLG2, respectively. The accessory subunits enhance DNA binding and promote processive DNA synthesis of the holoenzyme. Mutations in either POLG or POLG2 are linked to disease and adversely affect maintenance of the mitochondrial genome, resulting in depletion, deletions and/or point mutations in mtDNA. A homozygous mutation located at Chr17: 62492543G>A in POLG2, resulting in R182W substitution in p55, was previously identified to cause mtDNA depletion and fatal hepatic liver failure. Here we characterize this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol γ in vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203198PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114919PMC
February 2019

The C-terminal tail of the NEIL1 DNA glycosylase interacts with the human mitochondrial single-stranded DNA binding protein.

DNA Repair (Amst) 2018 05 6;65:11-19. Epub 2018 Mar 6.

University of South Alabama, Mitchell Cancer Institute, 1660 Springhill Avenue, Mobile, AL 36604, United States. Electronic address:

The 16.5 kb mitochondrial genome is subjected to damage from reactive oxygen species (ROS) generated in the cell during normal cellular metabolism and external sources such as ionizing radiation and ultraviolet light. ROS cause harmful damage to DNA bases that could result in mutagenesis and various diseases, if not properly repaired. The base excision repair (BER) pathway is the primary pathway involved in maintaining the integrity of mtDNA. Several enzymes that partake in BER within the nucleus have also been identified in the mitochondria. The nei-like (NEIL) DNA glycosylases initiate BER by excising oxidized pyrimidine bases and others such as the ring-opened formamidopyrimidine and the hydantoin lesions. During BER, the NEIL enzymes interact with proteins that are involved with DNA replication and transcription. In the current manuscript, we detected NEIL1 in purified mitochondrial extracts from human cells and showed that NEIL1 interacts with the human mitochondrial single-stranded DNA binding protein (mtSSB) via its C-terminal tail using protein painting, far-western analysis, and gel-filtration chromatography. Finally, we scrutinized the NEIL1-mtSSB interaction in the presence and absence of a partial-duplex DNA substrate using a combination of multi-angle light scattering (MALS) and small-angle X-ray scattering (SAXS). The data indicate that NEIL1 and homotetrameric mtSSB form a larger ternary complex in presence of DNA, however, the tetrameric form of mtSSB gets disrupted by NEIL1 in the absence of DNA as revealed by the formation of a smaller NEIL1-mtSSB complex.
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http://dx.doi.org/10.1016/j.dnarep.2018.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5911420PMC
May 2018

DNA polymerase β: A missing link of the base excision repair machinery in mammalian mitochondria.

DNA Repair (Amst) 2017 12 28;60:77-88. Epub 2017 Oct 28.

Genome Integrity and Structural Biology Laboratory, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709, USA. Electronic address:

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability.
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http://dx.doi.org/10.1016/j.dnarep.2017.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919216PMC
December 2017

Complementation of aprataxin deficiency by base excision repair enzymes in mitochondrial extracts.

Nucleic Acids Res 2017 Sep;45(17):10079-10088

Genome Integrity and Structural Biology Laboratory, DNA Repair and Nucleic Acid Enzymology Group, National Institutes of Health, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.

Mitochondrial aprataxin (APTX) protects the mitochondrial genome from the consequence of ligase failure by removing the abortive ligation product, i.e. the 5'-adenylate (5'-AMP) group, during DNA replication and repair. In the absence of APTX activity, blocked base excision repair (BER) intermediates containing the 5'-AMP or 5'-adenylated-deoxyribose phosphate (5'-AMP-dRP) lesions may accumulate. In the current study, we examined DNA polymerase (pol) γ and pol β as possible complementing enzymes in the case of APTX deficiency. The activities of pol β lyase and FEN1 nucleotide excision were able to remove the 5'-AMP-dRP group in mitochondrial extracts from APTX-/- cells. However, the lyase activity of purified pol γ was weak against the 5'-AMP-dRP block in a model BER substrate, and this activity was not able to complement APTX deficiency in mitochondrial extracts from APTX-/-Pol β-/- cells. FEN1 also failed to provide excision of the 5'-adenylated BER intermediate in mitochondrial extracts. These results illustrate the potential role of pol β in complementing APTX deficiency in mitochondria.
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http://dx.doi.org/10.1093/nar/gkx654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622373PMC
September 2017

Synergistic Effects of the T251I and P587L Mitochondrial DNA Polymerase γ Disease Mutations.

J Biol Chem 2017 03 2;292(10):4198-4209. Epub 2017 Feb 2.

From the Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Human mitochondrial DNA (mtDNA) polymerase γ (Pol γ) is the only polymerase known to replicate the mitochondrial genome. The Pol γ holoenzyme consists of the p140 catalytic subunit (POLG) and the p55 homodimeric accessory subunit (POLG2), which enhances binding of Pol γ to DNA and promotes processivity of the holoenzyme. Mutations within impede maintenance of mtDNA and cause mitochondrial diseases. Two common mutations usually found in patients primarily with progressive external ophthalmoplegia generate T251I and P587L amino acid substitutions. To determine whether T251I or P587L is the primary pathogenic allele or whether both substitutions are required to cause disease, we overproduced and purified WT, T251I, P587L, and T251I + P587L double variant forms of recombinant Pol γ. Biochemical characterization of these variants revealed impaired DNA binding affinity, reduced thermostability, diminished exonuclease activity, defective catalytic activity, and compromised DNA processivity, even in the presence of the p55 accessory subunit. However, physical association with p55 was unperturbed, suggesting intersubunit affinities similar to WT. Notably, although the single mutants were similarly impaired, a dramatic synergistic effect was found for the double mutant across all parameters. In conclusion, our analyses suggest that individually both T251I and P587L substitutions functionally impair Pol γ, with greater pathogenicity predicted for the single P587L variant. Combining T251I and P587L induces extreme thermal lability and leads to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients.
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http://dx.doi.org/10.1074/jbc.M116.773341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354484PMC
March 2017

POLG2 deficiency causes adult-onset syndromic sensory neuropathy, ataxia and parkinsonism.

Ann Clin Transl Neurol 2017 01 16;4(1):4-14. Epub 2016 Nov 16.

Department of Molecular and Human Genetics Baylor College of Medicine Houston Texas.

Objective: Mitochondrial dysfunction plays a key role in the pathophysiology of neurodegenerative disorders such as ataxia and Parkinson's disease. We describe an extended Belgian pedigree where seven individuals presented with adult-onset cerebellar ataxia, axonal peripheral ataxic neuropathy, and tremor, in variable combination with parkinsonism, seizures, cognitive decline, and ophthalmoplegia. We sought to identify the underlying molecular etiology and characterize the mitochondrial pathophysiology of this neurological syndrome.

Methods: Clinical, neurophysiological, and neuroradiological evaluations were conducted. Patient muscle and cultured fibroblasts underwent extensive analyses to assess mitochondrial function. Genetic studies including genome-wide sequencing were conducted.

Results: Hallmarks of mitochondrial dysfunction were present in patients' tissues including ultrastructural anomalies of mitochondria, mosaic cytochrome oxidase deficiency, and multiple mtDNA deletions. We identified a splice acceptor variant in c.970-1G>C, segregating with disease in this family and associated with a concomitant decrease in levels of POLG2 protein in patient cells.

Interpretation: This work extends the clinical spectrum of POLG2 deficiency to include an overwhelming, adult-onset neurological syndrome that includes cerebellar syndrome, peripheral neuropathy, tremor, and parkinsonism. We therefore suggest to include sequencing in the evaluation of ataxia and sensory neuropathy in adults, especially when it is accompanied by tremor or parkinsonism with white matter disease. The demonstration that deletions of mtDNA resulting from autosomal-dominant variant lead to a monogenic neurodegenerative multicomponent syndrome provides further evidence for a major role of mitochondrial dysfunction in the pathomechanism of nonsyndromic forms of the component neurodegenerative disorders.
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http://dx.doi.org/10.1002/acn3.361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221457PMC
January 2017

DNA polymerases in the mitochondria: A critical review of the evidence.

Front Biosci (Landmark Ed) 2017 01 1;22:692-709. Epub 2017 Jan 1.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, 111 T.W. Alexander Dr., Bldg. 101, Rm. E316, Research Triangle Park, NC 27709,

Since 1970, the DNA polymerase gamma (PolG) has been known to be the DNA polymerase responsible for replication and repair of mitochondrial DNA, and until recently it was generally accepted that this was the only polymerase present in mitochondria. However, recent data has challenged that opinion, as several polymerases are now proposed to have activity in mitochondria. To date, their exact role of these other DNA polymerases is unclear and the amount of evidence supporting their role in mitochondria varies greatly. Further complicating matters, no universally accepted standards have been set for definitive proof of the mitochondrial localization of a protein. To gain an appreciation of these newly proposed DNA polymerases in the mitochondria, we review the evidence and standards needed to establish the role of a polymerase in the mitochondria. Employing PolG as an example, we established a list of criteria necessary to verify the existence and function of new mitochondrial proteins. We then apply this criteria towards several other putative mitochondrial polymerases. While there is still a lot left to be done in this exciting new direction, it is clear that PolG is not acting alone in mitochondria, opening new doors for potential replication and repair mechanisms.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485829PMC
http://dx.doi.org/10.2741/4510DOI Listing
January 2017

DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides.

Nucleic Acids Res 2016 Nov 2;44(19):9381-9392. Epub 2016 Sep 2.

Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA

DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. Here, we demonstrate that Polθ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as Polθ. We further find that Polθ is inhibited after multiple dxGMP incorporation events, and that Polθ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for Polθ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of Polθ activity.
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http://dx.doi.org/10.1093/nar/gkw721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100566PMC
November 2016

Whole exome sequencing identifies a homozygous POLG2 missense variant in an infant with fulminant hepatic failure and mitochondrial DNA depletion.

Eur J Med Genet 2016 Oct 31;59(10):540-5. Epub 2016 Aug 31.

Department of Pathology and Cell Biology, Columbia University, 630 W, 168th Street, New York, NY 10032, USA; Division of Personalized Genomic Medicine, Department of Pathology and Cell Biology, Columbia University Medical Center, USA. Electronic address:

Mitochondrial DNA (mtDNA) depletion syndrome manifests as diverse early-onset diseases that affect skeletal muscle, brain and liver function. Mutations in several nuclear DNA-encoded genes cause mtDNA depletion. We report on a patient, a 3-month-old boy who presented with hepatic failure, and was found to have severe mtDNA depletion in liver and muscle. Whole-exome sequencing identified a homozygous missense variant (c.544C > T, p.R182W) in the accessory subunit of mitochondrial DNA polymerase gamma (POLG2), which is required for mitochondrial DNA replication. This variant is predicted to disrupt a critical region needed for homodimerization of the POLG2 protein and cause loss of processive DNA synthesis. Both parents were phenotypically normal and heterozygous for this variant. Heterozygous mutations in POLG2 were previously associated with progressive external ophthalmoplegia and mtDNA deletions. This is the first report of a patient with a homozygous mutation in POLG2 and with a clinical presentation of severe hepatic failure and mitochondrial depletion.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045816PMC
http://dx.doi.org/10.1016/j.ejmg.2016.08.012DOI Listing
October 2016

Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases.

Ageing Res Rev 2017 Jan 30;33:89-104. Epub 2016 Apr 30.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address:

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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http://dx.doi.org/10.1016/j.arr.2016.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5086445PMC
January 2017

Human mitochondrial DNA replication machinery and disease.

Curr Opin Genet Dev 2016 06 9;38:52-62. Epub 2016 Apr 9.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, United States. Electronic address:

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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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055853PMC
http://dx.doi.org/10.1016/j.gde.2016.03.005DOI Listing
June 2016

Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ.

Methods Mol Biol 2016 ;1351:19-26

Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Dr, Building 101, Rm E316, Research Triangle Park, NC, 27709, USA.

Mitochondrial DNA is replicated by the nuclear-encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand cross-links from chemotherapy agents. Although many of these lesions block DNA replication, pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis.
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http://dx.doi.org/10.1007/978-1-4939-3040-1_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000860PMC
September 2016

POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.

Hum Mol Genet 2015 Sep 29;24(18):5184-97. Epub 2015 Jun 29.

Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA

Human mitochondrial DNA (mtDNA) is replicated and repaired by the mtDNA polymerase gamma, polγ. Polγ is composed of three subunits encoded by two nuclear genes: (1) POLG codes for the 140-kilodalton (kDa) catalytic subunit, p140 and (2) POLG2 encodes the ∼110-kDa homodimeric accessory subunit, p55. Specific mutations are associated with POLG- or POLG2-related disorders. During DNA replication the p55 accessory subunit binds to p140 and increases processivity by preventing polγ's dissociation from the template. To date, studies have demonstrated that homodimeric p55 disease variants are deficient in the ability to stimulate p140; however, all patients currently identified with POLG2-related disorders are heterozygotes. In these patients, we expect p55 to occur as 25% wild-type (WT) homodimers, 25% variant homodimers and 50% heterodimers. We report the development of a tandem affinity strategy to isolate p55 heterodimers. The WT/G451E p55 heterodimer impairs polγ function in vitro, demonstrating that the POLG2 c.1352G>A/p.G451E mutation encodes a dominant negative protein. To analyze the subcellular consequence of disease mutations in HEK293 cells, we designed plasmids encoding p55 disease variants tagged with green fluorescent protein (GFP). P205R and L475DfsX2 p55 variants exhibit irregular diffuse mitochondrial fluorescence and unlike WT p55, they fail to form distinct puncta associated with mtDNA nucleoids. Furthermore, homogenous preparations of P205R and L475DfsX2 p55 form aberrant reducible multimers. We predict that abnormal protein folding or aggregation or both contribute to the pathophysiology of these disorders. Examination of mitochondrial bioenergetics in stable cell lines overexpressing GFP-tagged p55 variants revealed impaired mitochondrial reserve capacity.
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http://dx.doi.org/10.1093/hmg/ddv240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550827PMC
September 2015

Mitochondrial Disease Sequence Data Resource (MSeqDR): a global grass-roots consortium to facilitate deposition, curation, annotation, and integrated analysis of genomic data for the mitochondrial disease clinical and research communities.

Mol Genet Metab 2015 Mar 4;114(3):388-96. Epub 2014 Dec 4.

Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA. Electronic address:

Success rates for genomic analyses of highly heterogeneous disorders can be greatly improved if a large cohort of patient data is assembled to enhance collective capabilities for accurate sequence variant annotation, analysis, and interpretation. Indeed, molecular diagnostics requires the establishment of robust data resources to enable data sharing that informs accurate understanding of genes, variants, and phenotypes. The "Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium" is a grass-roots effort facilitated by the United Mitochondrial Disease Foundation to identify and prioritize specific genomic data analysis needs of the global mitochondrial disease clinical and research community. A central Web portal (https://mseqdr.org) facilitates the coherent compilation, organization, annotation, and analysis of sequence data from both nuclear and mitochondrial genomes of individuals and families with suspected mitochondrial disease. This Web portal provides users with a flexible and expandable suite of resources to enable variant-, gene-, and exome-level sequence analysis in a secure, Web-based, and user-friendly fashion. Users can also elect to share data with other MSeqDR Consortium members, or even the general public, either by custom annotation tracks or through the use of a convenient distributed annotation system (DAS) mechanism. A range of data visualization and analysis tools are provided to facilitate user interrogation and understanding of genomic, and ultimately phenotypic, data of relevance to mitochondrial biology and disease. Currently available tools for nuclear and mitochondrial gene analyses include an MSeqDR GBrowse instance that hosts optimized mitochondrial disease and mitochondrial DNA (mtDNA) specific annotation tracks, as well as an MSeqDR locus-specific database (LSDB) that curates variant data on more than 1300 genes that have been implicated in mitochondrial disease and/or encode mitochondria-localized proteins. MSeqDR is integrated with a diverse array of mtDNA data analysis tools that are both freestanding and incorporated into an online exome-level dataset curation and analysis resource (GEM.app) that is being optimized to support needs of the MSeqDR community. In addition, MSeqDR supports mitochondrial disease phenotyping and ontology tools, and provides variant pathogenicity assessment features that enable community review, feedback, and integration with the public ClinVar variant annotation resource. A centralized Web-based informed consent process is being developed, with implementation of a Global Unique Identifier (GUID) system to integrate data deposited on a given individual from different sources. Community-based data deposition into MSeqDR has already begun. Future efforts will enhance capabilities to incorporate phenotypic data that enhance genomic data analyses. MSeqDR will fill the existing void in bioinformatics tools and centralized knowledge that are necessary to enable efficient nuclear and mtDNA genomic data interpretation by a range of shareholders across both clinical diagnostic and research settings. Ultimately, MSeqDR is focused on empowering the global mitochondrial disease community to better define and explore mitochondrial diseases.
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http://dx.doi.org/10.1016/j.ymgme.2014.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512182PMC
March 2015

MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

PLoS Genet 2014 Oct 23;10(10):e1004748. Epub 2014 Oct 23.

Mitochondrial DNA Replication Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, North Carolina, United States of America.

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities.
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http://dx.doi.org/10.1371/journal.pgen.1004748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207668PMC
October 2014

Mitochondria, energetics, epigenetics, and cellular responses to stress.

Environ Health Perspect 2014 Dec 15;122(12):1271-8. Epub 2014 Aug 15.

Division of Extramural Research and Training, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Research Triangle Park, North Carolina, USA.

Background: Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria-nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation.

Objectives: We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function.

Methods: The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25-26 March 2013. Here, we summarize key points and ideas emerging from this meeting.

Discussion: A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways.

Conclusions: Understanding mitochondria-cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to environmental stressors.
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http://dx.doi.org/10.1289/ehp.1408418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256704PMC
December 2014

Defects of mitochondrial DNA replication.

J Child Neurol 2014 Sep 30;29(9):1216-24. Epub 2014 Jun 30.

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

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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http://dx.doi.org/10.1177/0883073814537380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146710PMC
September 2014

Mitochondrial genome maintenance in health and disease.

DNA Repair (Amst) 2014 Jul 26;19:190-8. Epub 2014 Apr 26.

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, Durham, NC 27709, USA.

Human mitochondria harbor an essential, high copy number, 16,569 base pair, circular DNA genome that encodes 13 gene products required for electron transport and oxidative phosphorylation. Mutation of this genome can compromise cellular respiration, ultimately resulting in a variety of progressive metabolic diseases collectively known as 'mitochondrial diseases'. Mutagenesis of mtDNA and the persistence of mtDNA mutations in cells and tissues is a complex topic, involving the interplay of DNA replication, DNA damage and repair, purifying selection, organelle dynamics, mitophagy, and aging. We briefly review these general elements that affect maintenance of mtDNA, and we focus on nuclear genes encoding the mtDNA replication machinery that can perturb the genetic integrity of the mitochondrial genome.
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http://dx.doi.org/10.1016/j.dnarep.2014.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075028PMC
July 2014

The exonuclease activity of the yeast mitochondrial DNA polymerase γ suppresses mitochondrial DNA deletions between short direct repeats in Saccharomyces cerevisiae.

Genetics 2013 Jun 15;194(2):519-22. Epub 2013 Apr 15.

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

The importance of mitochondrial DNA (mtDNA) deletions in the progeroid phenotype of exonuclease-deficient DNA polymerase γ mice has been intensely debated. We show that disruption of Mip1 exonuclease activity increases mtDNA deletions 160-fold, whereas disease-associated polymerase variants were mostly unaffected, suggesting that exonuclease activity is vital to avoid deletions during mtDNA replication.
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http://dx.doi.org/10.1534/genetics.113.150920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664861PMC
June 2013
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