Skip to main content
SpringerLink
Log in

Assay of mtDNA Polymerase γ from Human Tissues

  • Protocol
Mitochondrial DNA

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 197))

Abstract

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 (1–3). 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 (5–7). 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.

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  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.

    Article  CAS  Google Scholar 

  2. Shadel, G. S. and Clayton, D. A. (1997) Mitochondrial DNA maintenance in vertebrates. Annu. Rev. Biochem. 66, 409–435.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  8. Fry, M. and Loeb, L. A. (1986) Animal Cell DNA Polymerases, CRC, Boca Raton, FL, pp. 91–101.

    Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  10. De Vivo, D. C. (1993) The expanding clinical spectrum of mitochondrial diseases. Brain Dev. 15, 1–22.

    Article  PubMed  CAS  Google Scholar 

  11. Kerr, D. S. (1997) Protean manifestations of mitochondrial diseases: aminireview. J. Pediatr. Hematol./Oncol. 19, 279–286.

    Article  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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. 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. 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. 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.

    Article  PubMed  CAS  Google Scholar 

  23. Sheperd, D. and Garland, P. B. (1969) Citrate synthetase from rat liver. Methods Enzymol. 13, 11–16.

    Article  Google Scholar 

  24. Cornish-Bowden, A. (1995) Fundamentals of Enzyme Kinetics, rev. ed., Portland Press, London, pp. 30–37.

    Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Mitochondrial and Metabolic Disease Center, Department of Medicine, University of California, San Diego, School of Medicine, San Diego, CA

    Robert K. Naviaux

Authors
  1. Robert K. Naviaux
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

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

    William C. Copeland

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Naviaux, R.K. (2002). Assay of mtDNA Polymerase γ from Human Tissues. In: Copeland, W.C. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 197. Humana Press. https://doi.org/10.1385/1-59259-284-8:259

Download citation

  • DOI: https://doi.org/10.1385/1-59259-284-8:259

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-972-8

  • Online ISBN: 978-1-59259-284-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation