Abstract
The replicative mitochondrial DNA (mtDNA) helicase is essential for mtDNA replication and maintenance of the mitochondrial genome. Despite substantial advances that have been made in its characterization, there is still much to be understood about the functional roles of its domains and its interactions with the other components of the minimal mitochondrial DNA replisome. Critical to achieving this is the ability to isolate the enzyme in a stable, active form. In this chapter we describe a modified, streamlined purification strategy for recombinant forms of the enzyme. We also present assays to assess its helix unwinding activity and the stimulatory effects of the mitochondrial single-stranded DNA-binding protein (mtSSB). Finally, we describe a concentration/buffer exchange method that we have employed to achieve greater enzyme stability and appropriate conditions for biochemical and biophysical studies.
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References
Spelbrink J, Li F, Tiranti V, Nikali K, Yuan Q, Tariq M, Wanrooij S, Garrido N, Comi G, Morandi L, Santoro L, Toscano A, Fabrizi G, Somer H, Croxen R, Beeson D, Poulton J, Suomalainen A, Jacobs H, Zeviani M, Larsson C (2001) Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 28:223–231
Sanchez-Martinez A, Calleja M, Peralta S, Matsushima Y, Hernandez-Sierra R, Whitworth A, Kaguni L, Garesse R (2012) Modeling pathogenic mutations of human twinkle in Drosophila suggests an apoptosis role in response to mitochondrial defects. PLoS One 7:e43954
Matsushima Y, Kaguni L (2007) Differential phenotypes of active site and human autosomal dominant progressive external ophthalmoplegia mutations in Drosophila mitochondrial DNA helicase expressed in Schneider cells. J Biol Chem 282:9436–9444
Korhonen J, Gaspari M, Falkenberg M (2003) TWINKLE has 5′ → 3′ DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J Biol Chem 377:691–705
Oliveira M, Kaguni L (2010) Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein. PLoS One 5:e15379
Oliveira M, Kaguni L (2011) Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells. J Biol Chem 286:40649–40658
Ziebarth T, Farr C, Kaguni L (2007) Modular architecture of the hexameric human mitochondrial DNA helicase. J Mol Biol 367:1382–1391
Copeland W (2012) Defects in mitochondrial DNA replication and human disease. Crit Rev Biochem Mol Biol 47:64–74
Matsushima Y, Kaguni L (2009) Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase. Biochim Biophys Acta 1787:290–295
Holmlund T, Farge G, Pande V, Korhonen J, Nilsson L, Falkenberg M (2009) Structure-function defects of the twinkle amino-terminal region in progressive external ophthalmoplegia. Biochim Biophys Acta 1792:132–139
Ilyina T, Gorbalenya A, Koonin E (1992) Organization and evolution of bacterial and bacteriophage primase-helicase systems. J Mol Evol 34:351–357
Shutt T, Gray M (2006) Twinkle, the mitochondrial replicative DNA helicase, is widespread in the eukaryotic radiation and may also be the mitochondrial DNA primase in most eukaryotes. J Mol Evol 62:588–599
Ziebarth T, Kaguni L (2009) Purification strategy for recombinant forms of the human mitochondrial DNA helicase. Methods Mol Biol 554:121–126
Ziebarth T, Gonzalez-Soltero R, Makowska-Grzyska M, Nunez-Ramirez R, Carazo J, Kaguni L (2010) Dynamic effects of cofactors and DNA on the oligomeric state of human mitochondrial DNA helicase. J Biol Chem 285:4639–4647
Acknowledgements
This work was supported by grant GM45295 from the National Institutes of Health to L.S.K.
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Rosado-Ruiz, F.A., So, M., Kaguni, L.S. (2016). Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase. In: McKenzie, M. (eds) Mitochondrial DNA. Methods in Molecular Biology, vol 1351. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3040-1_14
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DOI: https://doi.org/10.1007/978-1-4939-3040-1_14
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3039-5
Online ISBN: 978-1-4939-3040-1
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