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Mitochondrial DNA Oxidative Damage and Mutagenesis in Saccharomyces cerevisiae

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Mitochondrial DNA

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 554))

Abstract

Mutation of human mitochondrial DNA (mtDNA) has been linked to maternally inherited neuromuscular disorders and is implicated in more common diseases such as cancer, diabetes, and Parkinson’s disease. Mutations in mtDNA also accumulate with age and are therefore believed to contribute to aging and age-related pathology. Housed within the mitochondrial matrix, mtDNA encodes several of the proteins involved in the production of ATP via the process of oxidative phosphorylation, which involves the flow of high-energy electrons through the electron transport chain (ETC). Because of its proximity to the ETC, mtDNA is highly vulnerable to oxidative damage mediated by reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals that are constantly produced by this system. Therefore, it is important to be able to measure oxidative mtDNA damage under normal physiologic conditions and during environmental or disease-associated stress. The budding yeast, Saccharomyces cerevisiae, is a facile and informative model system in which to study such mtDNA oxidative damage because it is a unicellular eukaryotic facultative anaerobe that is conditionally dependent on mitochondrial oxidative phosphorylation for viability. Here, we describe methods for quantifying oxidative mtDNA damage and mutagenesis in S. cerevisiae, several of which could be applied to the development of similar assays in mammalian cells and tissues. These methods include measuring the number of point mutations that occur in mtDNA with the erythromycin resistance assay, quantifying the amount of oxidative DNA damage utilizing a modified Southern blot assay, and measuring mtDNA integrity with the “petite induction” assay.

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Author information

Authors and Affiliations

  1. Emory University School of Medicine, Atlanta, GA, USA

    Lyra M. Griffiths & Paul W. Doetsch

  2. New York University School of Medicine, New York, NY, USA

    Nicole A. Doudican

  3. Yale University School of Medicine, New Haven, CT, USA

    Gerald S. Shadel

Authors
  1. Lyra M. Griffiths
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  2. Nicole A. Doudican
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  3. Gerald S. Shadel
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  4. Paul W. Doetsch
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Editor information

Editors and Affiliations

  1. Department of Biological Sciences, Brock University, 500 Glenridge Avenue, Cpy Catharines, ON, L2S 3A1, Canada

    Jeffrey A. Stuart

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Griffiths, L.M., Doudican, N.A., Shadel, G.S., Doetsch, P.W. (2009). Mitochondrial DNA Oxidative Damage and Mutagenesis in Saccharomyces cerevisiae . 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_17

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  • DOI: https://doi.org/10.1007/978-1-59745-521-3_17

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-60-2

  • Online ISBN: 978-1-59745-521-3

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