Abstract
The human mitochondrial genome encodes a variety of genes required for oxidative phosphorylation, and loss of these essential gene functions induces a multitude of severe metabolic disorders (1–4). Mitochondrial DNA (mtDNA) is replicated by the nuclear encoded DNA polymerase γ (5), and pol γ is the only replicative eukaryotic DNA polymerase to display sensitivity to a wide range of antiviral nucleotide analogs (6–12). Consequently, long-term treatment of patients with antiviral nucleotide analogs such as zidovudine (AZT), zalcitabine (ddC), and didanosine (ddI) induces a characteristic collection of mitochondrial dysfunctions that mimics mitochondrial genetic diseases (13–16). To aid investigations into the properties and subunit composition of this important enzyme, we present a simple biochemical assay to distinguish two forms of pol γ and a purification scheme to resolve them.
Keywords
- Catalytic Subunit
- Tissue Culture Cell
- Reverse Transcriptase Activity
- Accessory Subunit
- Reverse Transcriptase Assay
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H., Coulson, A. R., Drouin, J., et al. (1981) Sequence and organization of the human mitochondrial genome. Nature 290, 457–465.
Wallace, D. C. (1992) Diseases of the mitochondrial DNA. Annu. Rev. Biochem. 61, 1175–1212.
Luft, R. (1994) The development of mitochondrial medicine. Proc. Natl. Acad. Sci.USA 91, 8731–8738.
Wallace, D. C. (1999) Mitochondrial diseases in man and mouse. Science 283, 1482–1488.
Clayton, D. A. (1982) Replication of animal mitochondrial DNA. Cell 28, 693–705.
Kornberg, A. and Baker, T. A. (1992) DNA Replication, 2nd Ed., W.H. Freeman, New York.
Fry, M. and Loeb, L. A. (1986) Animal Cell DNA Polymerases, CRC, Boca Raton, FL.
Lewis, W., Meyer, R. R., Simpson, J. F., Colacino, J. M., and Perrino, F. W. (1994) Mammalian DNA polymerases alpha, beta, gamma, delta, and epsilon incorporate fialuridine (FIAU) monophosphate into DNA and are inhibited competitively by FIAU Triphosphate. Biochemistry 33, 14,620–14,624.
Wang, T. S.-F. (1991) Eukaryotic DNA polymerases. Annu. Rev. Biochem. 60, 513–552.
Cherrington, J. M., Allen, S. J., McKee, B. H., and Chen, M. S. (1994) Kinetic analysis of the interaction between the diphosphate of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, ddCTP, AZTTP, and FIAUTP with human DNA polymerases beta and gamma. Biochem. Pharmacol. 48, 1986–1988.
Faraj, A., Fowler, D. A., Bridges, E. G., and Sommadossi, J. P. (1994) Effects of 2?,3?-dideoxynucleosides on proliferation and differentiation of human pluripotent progenitors in liquid culture and their effects on mitochondrial DNA synthesis. Antimicrob. Agents Chemother. 38, 924–930.
Eriksson, S., Xu, B., and Clayton, D. A. (1995) Efficient incorporation of anti-HIV deoxynucleotides by recombinant yeast mitochondrial DNA polymerase. J. Biol. Chem. 270, 18,929–18,934.
Dalakas, M. C., Illa, I., Pezeshkpour, G. H., Laukaitis, J. P., Cohen, B., and Griffin, J. L. (1990) Mitochondrial myopathy caused by long-term zidovudine therapy [see comments]. N. Engl. J. Med. 322, 1098–1105.
Arnaudo, E., Dalakas, M., Shanske, S., Moraes, C. T., DiMauro, S., and Schon, E. A. (1991) Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 337, 508–510.
Peters, B. S., Winer, J., Landon, D. N., Stotter, A., and Pinching, A. J. (1993) Mitochondrial myopathy associated with chronic zidovudine therapy in AIDS. Q. J. Med. 86, 5–15.
Lewis, W. and Dalakas, M. C. (1995) Mitochondrial toxicity of antiviral drugs. Nat. Med. 1, 417–422.
Wernette, C. M. and Kaguni, L. S. (1986) A mitochondrial DNA polymerase from embryos of Drosophila melanogaster. Purification, subunit structure, and partial characterization. J. Biol. Chem. 261, 14,764–14,770.
Insdorf, N. F. and Bogenhagen, D. F. (1989) DNA polymerase gamma from Xenopus laevis. I. The identification of a high molecular weight catalytic subunit by a novel DNA polymerase photolabeling procedure. J. Biol. Chem. 264, 21,491–21,497.
Gray, H. and Wong, T. W. (1992) Purification and identification of subunit structure of the human mitochondrial DNA polymerase. J. Biol. Chem. 267, 5835–5841.
Foury, F. (1989) Cloning and sequencing of the nuclear gene MIP1 encoding the catalytic subunit of the yeast mitochondrial DNA polymerase. J. Biol. Chem. 264, 20,552–20,560.
Ropp, P. A. and Copeland, W. C. (1995) Characterization of a new DNA polymerase from Schizosaccharomyces pombe: a probable homologue of the Saccharomyces cerevisiae DNA polymerase gamma. Gene 165, 103–107.
Ropp, P. A. and Copeland, W. C. (1996) Cloning and characterization of the human mitochondrial DNA polymerase, DNA polymerase gamma. Genomics 36, 449–458.
Ye, F., Carrodeguas, J. A., and Bogenhagen, D. F. (1996) The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma. Nucleic Acids Res. 24, 1481–1488.
Lewis, D. L., Farr, C. L., Wang, Y., Lagina, A. T. R., and Kaguni, L. S. (1996) Catalytic subunit of mitochondrial DNA polymerase from Drosophila embryos. Cloning, bacterial overexpression, and biochemical characterization. J. Biol. Chem. 271, 23,389–23,394.
Kunkel, T. A. and Soni, A. (1988) Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma. J. Biol. Chem. 263, 4450–4459.
Kunkel, T. A. and Mosbaugh, D. W. (1989) Exonucleolytic proofreading by a mammalian DNA polymerase. Biochemistry 28, 988–995.
Insdorf, N. F. and Bogenhagen, D. F. (1989) DNA polymerase gamma from Xenopus laevis. II. A 3?? 5? exonuclease is tightly associated with the DNA polymerase activity. J. Biol. Chem. 264, 21,498–21,503.
Olson, M. W. and Kaguni, L. S. (1992) 3?? 5? exonuclease in Drosophila mitochondrial DNA polymerase. Substrate specificity and functional coordination of nucleotide polymerization and mispair hydrolysis. J. Biol. Chem. 267, 23,136–23,142.
Foury, F. and Vanderstraeten, S. (1992) Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity. EMBO J. 11, 2717–2726.
Spelbrink, J. N., Toivonen, J. M., Hakkaart, G. A., Kurkela, J. M., Cooper, H. M., Lehtinen, S. K., et al. (2000) In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells [in process citation]. J. Biol. Chem. 275, 24,818–24,828.
Zhang, D., Mott, J. L., Chang, S. W., Denniger, G., Feng, Z., and Zassenhaus, H. P. (2000) Construction of transgenic mice with tissue-specific acceleration of mitochondrial DNA mutagenesis. Genomics 69, 151–161.
Longley, M. J., Ropp, P. A., Lim, S. E., and Copeland, W. C. (1998) Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity. Biochemistry 37, 10,529–10,539.
Lim, S. E., Longley, M. J., and Copeland, W. C. (1999) The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance. J. Biol. Chem. 274, 38,197–38,203.
Mosbaugh, D. W. (1988) Purification and characterization of porcine liver DNA polymerase gamma: utilization of dUTP and dTTP during in vitro DNA synthesis. Nucleic Acids Res. 16, 5645–5659.
Bogenhagen, D. and Clayton, D. A. (1974) The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Quantitative isolation of mitochondrial deoxyribonucleic acid. J. Biol. Chem. 249, 7991–7995.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Longley, M.J., Copeland, W.C. (2002). Purification, Separation, and Identification of the Human mtDNA Polymerase With and Without Its Accessory Subunit. In: Copeland, W.C. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 197. Humana Press. https://doi.org/10.1385/1-59259-284-8:245
Download citation
DOI: https://doi.org/10.1385/1-59259-284-8:245
Publisher Name: Humana Press
Print ISBN: 978-0-89603-972-8
Online ISBN: 978-1-59259-284-5
eBook Packages: Springer Protocols