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Mitochondrial Base Excision Repair Assays

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 920)

Abstract

Mitochondrial DNA (mtDNA) is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to be particularly deleterious in post-mitotic cells, including neurons, and to play a critical role in the aging process and in a variety of diseases. Thus, efficient mtDNA repair is important for the maintenance of genomic integrity and a healthy life. The base excision repair (BER) mechanism was the first to be described in mitochondria, and consequently it is the best known. This chapter outlines protocols for isolating mitochondria from mammalian cells in culture and from rodent tissues including liver and brain. It also covers the isolation of synaptic mitochondria. BER takes place in four distinct steps, and protocols describing in vitro assays for measuring these enzymatic steps in lysates of isolated mitochondria are included.

Key words

Mitochondria mtDNA DNA repair Base excision repair 
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Notes

Acknowledgements

The authors gratefully acknowledge the financial support from the Velux Foundation (Danish Aging Research Center) and The Danish Research Councils to TS, and from CAM/UCM (CCG10-UCM/SAL-4798) to RG. We also thank the members of the Laboratory for DNA repair and Aging at Dep. of Molecular Biology and Genetics, Aarhus University and the members of the Laboratory of Molecular Gerontology at National Institutes of Health, NIH for longstanding collaborations.

References

  1. 1.
    Grollman AP, Moriya M (1993) Mutagenesis by 8-oxoguanine: an enemy within. Trends Genet 9:246–249PubMedCrossRefGoogle Scholar
  2. 2.
    Kavli B, Otterlei M, Slupphaug G, Krokan HE (2007) Uracil in DNA—general mutagen, but normal intermediate in acquired immunity. DNA Repair (Amst) 6:505–516CrossRefGoogle Scholar
  3. 3.
    Croteau DL, Bohr VA (1997) Repair of oxidative damage to nuclear and mitochondrial DNA in mammalian cells. J Biol Chem 272:25409–25412PubMedCrossRefGoogle Scholar
  4. 4.
    Hamilton ML, Guo Z, Fuller CD, Van Remmen H, Ward WF, Austad SN, Troyer DA, Thompson I, Richardson A (2001) A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA. Nucleic Acids Res 29:2117–2126PubMedCrossRefGoogle Scholar
  5. 5.
    Barja G, Herrero A (2000) Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals. FASEB J 14:312–318PubMedGoogle Scholar
  6. 6.
    Schon EA (2000) Mitochondrial genetics and disease. Trends Biochem Sci 25:555–560PubMedCrossRefGoogle Scholar
  7. 7.
    Beal MF (2005) Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 58:495–505PubMedCrossRefGoogle Scholar
  8. 8.
    Melov S (2004) Modeling mitochondrial function in aging neurons. Trends Neurosci 27:601–606PubMedCrossRefGoogle Scholar
  9. 9.
    Krishnan KJ, Reeve AK, Samuels DC, Chinnery PF, Blackwood JK, Taylor RW, Wanrooij S, Spelbrink JN, Lightowlers RN, Turnbull DM (2008) What causes mitochondrial DNA deletions in human cells? Nat Genet 40:275–279PubMedCrossRefGoogle Scholar
  10. 10.
    Weissman L, de Souza-Pinto NC, Stevnsner T, Bohr VA (2007) DNA repair, mitochondria, and neurodegeneration. Neuroscience 145:1318–1329PubMedCrossRefGoogle Scholar
  11. 11.
    Gredilla R, Bohr VA, Stevnsner T (2010) Mitochondrial DNA repair and association with aging—an update. Exp Gerontol 45:478–488PubMedCrossRefGoogle Scholar
  12. 12.
    Seeberg E, Eide L, Bjoras M (1995) The base excision repair pathway. Trends Biochem Sci 20:391–397PubMedCrossRefGoogle Scholar
  13. 13.
    Bohr VA (2002) DNA damage and its processing. Relation to human disease. J Inherit Metab Dis 25:215–222PubMedCrossRefGoogle Scholar
  14. 14.
    Bohr VA (2002) Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic Biol Med 32:804–812PubMedCrossRefGoogle Scholar
  15. 15.
    Stuart JA, Karahalil B, Hogue BA, Souza-Pinto NC, Bohr VA (2004) Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction. FASEB J 18:595–597PubMedGoogle Scholar
  16. 16.
    Karahalil B, Hogue BA, de Souza-Pinto NC, Bohr VA (2002) Base excision repair capacity in mitochondria and nuclei: tissue-specific variations. FASEB J 16:1895–1902PubMedCrossRefGoogle Scholar
  17. 17.
    Lai JC, Clark JB (1979) Preparation of synaptic and nonsynaptic mitochondria from mammalian brain. Methods Enzymol 55:51–60PubMedCrossRefGoogle Scholar
  18. 18.
    Kristian T, Hopkins IB, McKenna MC, Fiskum G (2006) Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation. J Neurosci Methods 152:136–143PubMedCrossRefGoogle Scholar
  19. 19.
    Brown MR, Sullivan PG, Geddes JW (2006) Synaptic mitochondria are more susceptible to Ca2+ overload than nonsynaptic mitochondria. J Biol Chem 281:11658–11668PubMedCrossRefGoogle Scholar
  20. 20.
    Schrimpf SP, Meskenaite V, Brunner E, Rutishauser D, Walther P, Eng J, Aebersold R, Sonderegger P (2005) Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry. Proteomics 5:2531–2541PubMedCrossRefGoogle Scholar
  21. 21.
    Gredilla R, Weissman L, Yang JL, Bohr VA, Stevnsner T (2010) Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer’s disease. Neurobiol Aging 33(4):694–707PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Department of PhysiologyFaculty of Medicine,Complutense UniversityMadridSpain
  2. 2.Department of Molecular Biology and GeneticsUniversity of AarhusAarhusDenmark

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