Assay of mtDNA Polymerase γ from Human Tissues

  • Robert K. Naviaux
Part of the Methods in Molecular Biology™ book series (MIMB, volume 197)


Mitochondrial DNA (mtDNA) replication is a complex process involving over 20 proteins organized along the inner mitochondrial membrane as a multienzyme complex called the mtDNA replisome, or replication factory (1, 2, 3). Figure 1 illustrates some of the protein components that participate in mitochondrial DNA replication. A principal component of the mtDNA replisome is the mtDNA polymerase γ. This enzyme is found as an αβ heterodimer and as an α monomer associated with at least four other unidentified cellular proteins (4). Both α- and β-subunits have been cloned (5, 6, 7). The α-subunit is catalytic and contains both the polymerase and the 3′ to 5′ proofreading exonuclease activities. It is 140,000 Daltons in size.
Fig. 1.

The mitochondrial DNA replisome. The actual geometry and subunit composition of the mtDNA replisome is not yet known. The proteins shown are those that have been shown to participate in mtDNA replication or repair.


Mitochondrial Protein Skeletal Muscle Mitochondrion Mitochondrial Matrix Protein Alpers Syndrome Suspected Mitochondrial Disease 
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  1. 1.
    Reddy, G. P. V. and Pardee, A. B. (1980) Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc. Natl. Acad. Sci. USA 77, 3312–3316.CrossRefGoogle Scholar
  2. 2.
    Shadel, G. S. and Clayton, D. A. (1997) Mitochondrial DNA maintenance in vertebrates. Annu. Rev. Biochem. 66, 409–435.PubMedCrossRefGoogle Scholar
  3. 3.
    Lemon, D. P. and Grossman, A. D. (1998) Localization of bacterial DNA polymerase: evidence for a factory model of replication. Science 282, 1516–1519.PubMedCrossRefGoogle Scholar
  4. 4.
    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 γ: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity. Biochemistry 37, 10,529–10,539.PubMedCrossRefGoogle Scholar
  5. 5.
    Ropp, P. A. and Copeland, W. C. (1996) Cloning and characterization of the human mitochondrial DNA polymerase, DNA polymerase γ. Genomics 36, 449–458.PubMedCrossRefGoogle Scholar
  6. 6.
    Lecrenier, N., Van Der Bruggen, P., and Foury, F. (1997) Mitochondrial DNA polymerases from yeast to man: a new family of polymerases. Gene 185, 147–152.PubMedCrossRefGoogle Scholar
  7. 7.
    Wang, Y., Farr C. L., and Kaguni, L. S. (1997) Accessory subunit of mitochondrial DNA polymerase from Drosophila embryos: cloning, molecular analysis, and association in the native enzyme. J. Biol. Chem. 272, 13,640–13,646.PubMedCrossRefGoogle Scholar
  8. 8.
    Fry, M. and Loeb, L. A. (1986) Animal Cell DNA Polymerases, CRC, Boca Raton, FL, pp. 91–101.Google Scholar
  9. 9.
    Lewis, W., Meyer, R. R., Simpson, J. F., Colacino, J. M., and Perrino, F. W. (1994) Mammalian DNA polymerases α, β, γ, δ, and ε incorporate fialuridine (FIAU) monophosphate into DNA and are inhibitied competitively by FIAU triphosphate. Biochemistry 33, 14,620–14,624.PubMedCrossRefGoogle Scholar
  10. 10.
    De Vivo, D. C. (1993) The expanding clinical spectrum of mitochondrial diseases. Brain Dev. 15, 1–22.PubMedCrossRefGoogle Scholar
  11. 11.
    Kerr, D. S. (1997) Protean manifestations of mitochondrial diseases: aminireview. J. Pediatr. Hematol./Oncol. 19, 279–286.CrossRefGoogle Scholar
  12. 12.
    Moraes, C.T., Shanske, S., Tritschler, H.-J., Aprille, J. R., Andreetta F., Bonilla, E., et al. (1991) mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am. J. Hum. Genet. 48, 492–501.PubMedGoogle Scholar
  13. 13.
    Ricci, E., Moraes, C. T., Servidei, S., Tonali, P., Bonilla, E., and DiMauro, S. (1992) Disorders associated with depletion of mitochondrial DNA. Brain Pathol. 2, 141–147.PubMedCrossRefGoogle Scholar
  14. 14.
    Poulton, J., Sewry, C., Potter, C. G., Bougeron, T., Chretien, D., Wijburg, F. A., et al. (1995) Variation in mitochondrial DNA levels in muscle from normal controls. Is depletion of mtDNA in patients with mitochondrial myopathy a distinct clinical syndrome? J. Inherit. Metabol. Dis. 18, 4–20.CrossRefGoogle Scholar
  15. 15.
    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. N. Engl. J. Med. 322, 1098–1105.PubMedCrossRefGoogle Scholar
  16. 16.
    Mckenzie, R., Fried, M. W., Sallie, R., Conjeevaram, H., Di Bisceglie, A. M., Park, Y., et al. (1995) Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B. N. Engl. J. Med. 333, 1099–1105.PubMedCrossRefGoogle Scholar
  17. 17.
    Naviaux, R. K., Nyhan, W. L., Barshop, B. A., Poulton, J., Markusic, D., Karpinski, N. C., et al. (1999) Mitochondrial DNA polymeraseγ deficiency and mitochondrial DNA depletion in a child with Alpers syndrome. Ann. Neurol. 45, 54–58.PubMedCrossRefGoogle Scholar
  18. 18.
    Naviaux, R. K., Markusic, D., Barshop, B., Nyhan, W. L., and Haas, R. H. (1999) Sensitive assay for mitochondrial DNA polymerase γ. Clin. Chem. 45, 1725–1733.PubMedGoogle Scholar
  19. 19.
    Attardi, G. M. and Chomyn, A., (eds.) (1995) Mitochondrial Biogenesis and Genetics, Part A, Methods in Enzymology Vol. 260, Academic, San Diego, CA.Google Scholar
  20. 20.
    Attardi, G. M. and Chomyn, A. (eds.) (1996) Mitochondrial Biogenesis and Genetics, Part B, Methods in Enzymology Vol. 264, Academic, San Diego, CA.Google Scholar
  21. 21.
    Taylor, R. W. and Turnbull, D. M. (1997) Laboratory diagnosis of mitochondrial disease, in Organelle Diseases (Applegarth, D. A., Dimmick, J. E., and Hall, J. G., eds.), Chapman & Hall, London, pp. 341–350.Google Scholar
  22. 22.
    Hatefi, Y., Jurtshuk, P., and Haavik, A. G. (1961) Studies on the electron transport system. 32. Respiratory control in beef heart mitochondria. Arch. Biochem. Biophys. 94, 148–155.PubMedCrossRefGoogle Scholar
  23. 23.
    Sheperd, D. and Garland, P. B. (1969) Citrate synthetase from rat liver. Methods Enzymol. 13, 11–16.CrossRefGoogle Scholar
  24. 24.
    Cornish-Bowden, A. (1995) Fundamentals of Enzyme Kinetics, rev. ed., Portland Press, London, pp. 30–37.Google Scholar
  25. 25.
    Copeland, W. C., Chen, M. S., and Wang, T. S. F. (1992) Human DNA polymerases a and β are able to incorporate anti-HIV deoxynucleotides into DNA. J. Biol. Chem. 267, 21,459–21,464.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Robert K. Naviaux
    • 1
  1. 1.Mitochondrial and Metabolic Disease Center, Department of MedicineUniversity of California, San Diego, School of MedicineSan Diego

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