Abstract
The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for ATP production. Disruption of mitochondrial energy metabolism can occur by genetic and biochemical mechanisms involving nuclear-encoded proteins that are required at the mitochondrial DNA replication fork, which often leads to human disorders and to animal lethality during development. DNA polymerase γ (pol γ), the mitochondrial replicase, and the mitochondrial single-stranded DNA-binding protein (mtSSB) have been the focus of study in our lab for a number of years. Here we describe the purification strategies that we developed for obtaining the recombinant forms of pol γ and mtSSB from both Drosophila melanogaster and humans. Despite the fact that similar approaches can be used for purifying the homologous proteins, we have observed that there are differences in the behavior of the proteins in some specific steps that may reflect differences in their structural and biochemical properties. Their purification in homogeneous, active form represents the first step toward our long-term goal to understand their biochemistry, biology, and functions at the mitochondrial DNA replication fork.
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References
Gray, M. W., Burger, G., and Lang, B. F. (1999) Mitochondrial evolution. Science 283, 1476–1481.
Kaguni, L.S. (2004) DNA polymerase gamma, the mitochondrial replicase. Annu. Rev. Biochem. 73, 293–320.
Korhonen, J. A., Pham, X. H., Pellegrini, M., and Falkenberg, M. (2004) Reconstitution of a minimal mtDNA replisome in vitro. EMBO J. 23, 2423–2429.
Fan, L., Kim, S., Farr, C. L., Schaefer, K. T., Randolph, K. M., Tainer, J. A., and Kaguni, L. S. (2006) A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase. J. Mol. Biol. 358, 1229–1243.
Fan, L., and Kaguni, L. S. (2001) Multiple regions of subunit interaction in Drosophila mitochondrial DNA polymerase: three functional domains in the accessory subunit. Biochemistry 40, 4780–4791.
Yakubovskaya, E., Chen, Z., Carrodeguas, J. A., Kisker, C., and Bogenhagen, D. F. (2006) Functional human mitochondrial DNA polymerase γ forms a heterotrimer. J. Biol. Chem. 281, 374–382.
Longley, M. J., Graziewicz, M. A., Bienstock, R. J., and Copeland, W. C. (2005) Consequences of mutations in human DNA polymerase γ. Gene 354, 125–131.
Luoma, P. T., Luo, N., Löscher, W. N., Farr, C. L., Horvath, R., Wanschitz, J., Kiechl, S., Kaguni, L. S., and Suomalainen, A. (2005) Functional defects due to spacer-region mutations of human mitochondrial DNA polymerase in a family with an ataxia-myopathy syndrome. Hum. Mol. Genet. 14, 1907–1920.
Iyengar, B., Roote, J., and Campos, A. R. (1999) The tamas gene, identified as a mutation that disrupts larval behavior in Drosophila melanogaster, codes for the mitochondrial DNA polymerase catalytic subunit (DNApol- γ125). Genetics 153, 1809–1824.
Luo, N., and Kaguni, L. S. (2005) Mutations in the spacer region of Drosophila mitochondrial DNA polymerase affect DNA binding, processivity, and the balance between pol and exo function. J. Biol. Chem. 280, 2491–2497.
Iyengar, B., Luo, N., Farr, C. L., Kaguni, L. S., and Campos, A. R. (2002) The accessory subunit of DNA polymerase γ is essential for mitochondrial DNA maintenance and development in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 99, 4483–4488.
Curth, U., Urbanke, C., Greipel, J., Gerberding, H., Tiranti, V., and Zeviani, M. (1994) Single-stranded DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties. Eur. J. Biochem. 221, 435–443.
Thommes, P., Farr, C. L., Marton, R. F., Kaguni, L. S., and Cotterill, S. (1995) Mitochondrial single-stranded DNA-binding protein from Drosophila embryos. Physical and biochemical characterization. J. Biol. Chem. 270, 21137–21143.
Farr, C. L., Wang, Y., and Kaguni, L. S. (1999) Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein: template-primer DNA binding and initiation and elongation of DNA strand synthesis. J. Biol. Chem. 274, 14779–14785.
Korhonen, J. A., Gaspari, M., and Falkenberg, M. (2003) TWINKLE has 5’→3’ DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J. Biol. Chem. 278, 48627–48632.
Maier, D., Farr, C. L., Poeck, B., Alahari, A., Vogel, M., Fischer, S., Kaguni, L. S., and Schneuwly, S. (2001) Mitochondrial single-stranded DNA-binding protein is required for mitochondrial DNA replication and development in Drosophila melanogaster. Mol. Biol. Cell 12, 821–830.
Fernández-Moreno, M. A., Farr, C. L., Kaguni, L. S., and Garesse, R. (2006) Drosophila melanogaster as a model system to study mitochondrial function. Methods Mol. Biol. 372, 33–49.
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, 14764–14770.
Wang, Y., and Kaguni, L. S. (1999) Baculovirus expression reconstitutes Drosophila mitochondrial DNA polymerase. J. Biol. Chem. 274, 28972–28977.
Farr, C. L., and Kaguni, L. S. (2002a) Purification strategies for Drosophila mtDNA replication proteins in native and recombinant form. DNA polymerase γ. Methods Mol. Biol. 197, 273–283.
Farr, C. L., and Kaguni, L. S. (2002b) Purification strategies for Drosophila mtDNA replication proteins in native and recombinant form. Mitochondrial single-stranded DNA-binding protein. Methods Mol. Biol. 197, 285–294.
Ziebarth, T. D., Farr, C. L., and Kaguni, L. S. (2007) Modular architecture of the hexameric human mitochondrial DNA helicase. J. Mol. Biol. 367, 1382–1391.
Fansler, B. S., and Loeb, L. A. (1974) Sea urchin nuclear DNA polymerase. Methods Enzymol. 29, 53–70.
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Oliveira, M.T., Kaguni, L.S. (2009). Comparative Purification Strategies for Drosophila and Human Mitochondrial DNA Replication Proteins: DNA Polymerase γ and Mitochondrial Single-Stranded DNA-Binding Protein. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_3
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DOI: https://doi.org/10.1007/978-1-59745-521-3_3
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Print ISBN: 978-1-934115-60-2
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