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Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells

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Book cover DNA Repair Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 314))

Abstract

In this chapter, we describe a gene-specific quantitative polymerase chain reaction (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been extensively used to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins, and has proved particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA (mtDNA) damage. QPCR can be used to quantify the formation of DNA damage, as well as the kinetics of damage removal. One of the main strengths of the assay is that it permits monitoring the integrity of mtDNA directly from total cellular DNA without the need for isolating mitochondria, or a separate step of mtDNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol for the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.

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References

  1. Ponti, M., Forrow, S. M., Souhami, R. L., D’Incalci, M., and Hartley, J. A. (1991) Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase. Nucleic Acids Res. 19, 2929–2933.

    Article  PubMed  CAS  Google Scholar 

  2. Jennerwein, M. M. and Eastman, A. (1991) A polymerase chain reaction-based method to detect cisplatin adducts in specific genes. Nucleic Acids Res. 19, 6209–6214.

    Article  PubMed  CAS  Google Scholar 

  3. Kalinowski, D., Illenye, S., and Van Houten, B. (1992) Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay. Nucleic Acids Res. 20, 3485–3494.

    Article  PubMed  CAS  Google Scholar 

  4. Van Houten, B., Cheng, S., and Chen, Y. (2000) Measuring DNA damage and repair in human genes using quantitative amplification of long targets from nanogram quantities of DNA. Mutat. Res. 460, 81–94.

    PubMed  Google Scholar 

  5. Van Houten, B., Chen, Y., Nicklas, J.A., Rainville, I.R., and O’Neill, J.P. (1998) Development of long PCR techniques to analyze deletion mutations of the human hprt gene. Mutat. Res. 403, 171–175.

    PubMed  Google Scholar 

  6. Ayala-Torres, S., Chen, Y., Svoboda, T., Rosenblatt, J., and Van Houten, B. (2000) Analysis of gene-specific DNA damage and repair using quantitative PCR. Methods 22, 135–147.

    Article  PubMed  CAS  Google Scholar 

  7. Yakes, F. M. and Van Houten, B. (1997) Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl. Acad. Sci. USA 94, 514–519.

    Article  PubMed  CAS  Google Scholar 

  8. Mandavilli, B. S., Ali, S. F., and Van Houten, B. (2000) DNA damage in brain mitochondria caused by aging and MPTP treatment. Brain Res. 885, 45–52.

    Article  PubMed  CAS  Google Scholar 

  9. Moon S. K., Thompson L. J., Madamanchi, N., et al. (2001) Aging, oxidative stress, and proliferative capacity in cultured mouse aortic smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. 280, 2779–2788.

    Google Scholar 

  10. Denissenko, M. F., Cahill, J., Koudriakova, T. B., Gerber, N., and Pfeifer, G. P. (1999) Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems. Mutat. Res. 425, 205–211.

    PubMed  CAS  Google Scholar 

  11. Ballinger, S. W., Patterson, C., Knight-Lozano, C. A., et al. (2002) Mitochondrial integrity and function in atherogenesis. Circulation 106, 544–549.

    Article  PubMed  CAS  Google Scholar 

  12. Jin, G. F., Hurst, J. S., and Godley, B. F. (2001) Rod outer segments mediate mitochondrial DNA damage and apoptosis in human retinal pigment epithelium. Curr. Eye Res. 23, 11–19.

    Article  PubMed  CAS  Google Scholar 

  13. Sawyer, D. E., Mercer, B. G., Wiklendt, A. M., and Aitken, R. J. (2003) Quantitative analysis of gene-specific DNA damage in human spermatozoa. Mutat. Res. 529, 21–34.

    PubMed  CAS  Google Scholar 

  14. Santos, J. H., Hunakova, L., Chen, Y., Bortner, C., and Van Houten, B. (2003) Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death. J. Biol. Chem. 278, 1728–1734.

    Article  PubMed  CAS  Google Scholar 

  15. Yanez, J. A., Teng, X. W., Roupe, K. A., Fariss, M. W., and Davies, N. M. (2003) Chemotherapy induced gastrointestinal toxicity in rats: involvement of mitochondrial DNA, gastrointestinal permeability and cyclooxygenase-2. J. Pharmacol. Pharmaceut. Sci. 6, 308–314.

    CAS  Google Scholar 

  16. O’Brien, T., Xu, J., and Patierno S. R. (2001) Effects of glutathione on chromium-induced crosslinking and DNA polymerase arrest. Mol. Cell Biochem. 222, 173–182.

    Article  CAS  Google Scholar 

  17. Chandrasekhar, D. and Van Houten, B. (1994) High resolution mapping of UV-induced photoproducts in the E. coli IacI gene: inefficient repair in the nontranscribed strand correlates with high mutation frequency. J. Mol. Biol. 238, 319–332.

    Article  PubMed  CAS  Google Scholar 

  18. Yakes, F. M., Chen, Y., and Van Houten, B. (1996) PCR-based assays for the detection and quantitation of DNA damage and repair, in Technologies for Detection of DNA Damage and Mutations, (Pfeifer, G. P., ed.). Plenum Press, New York, NY, pp. 171–184.

    Google Scholar 

  19. Salazar, J. J. and Van Houten, B. (1997) Preferential mitochondrial DNA injury caused by glucose oxidase as a steady generator for hydrogen peroxide in human fibroblasts. Mutat Res. 385, 139–149.

    PubMed  CAS  Google Scholar 

  20. Chen, K. H., Srivastava, D. K., Yakes, F. M., et al. (1998) Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines. Nucleic Acids Res. 26, 2001–2007.

    Article  PubMed  CAS  Google Scholar 

  21. Horton, J. K., Roy, G., Piper, J. T., et al. (1999) Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione s-transferase μ. Biochem. Pharmacol. 58, 693–702.

    Article  PubMed  CAS  Google Scholar 

  22. Deng, G., Su, J. H., Ivins, K. J., Van Houten, B., and Cottman, C. (1999) Bcl-2 facilitates recovery from DNA damage after oxidative stress. Experimental Neurol. 159, 309–318.

    Article  CAS  Google Scholar 

  23. Ballinger, S. W., Patterson, C., Yan, C. N., et al. (2000) Hydrogen peroxide-and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ. Res. 786, 960–966.

    Google Scholar 

  24. Chandrasekhar, D. and Van Houten, B. (2000) In vivo formation and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level in E. coli. Mutat. Res. 450, 19–40.

    PubMed  CAS  Google Scholar 

  25. Sobol, R. W., Watson, D. E., Nakamura, J., et al. (2002) Mutator phenotype associated with a gene-environment interaction: effect of base excision repair deficiency and methylation-induced genotoxic stress. Proc. Natl. Acad. Sci. USA 99, 6860–6865.

    Article  PubMed  CAS  Google Scholar 

  26. Wallace, D. C. (1999) Mitochondrial diseases in man and mouse. Science 283, 1482–1488.

    Article  PubMed  CAS  Google Scholar 

  27. DiMauro, S. and Schon, E. A. (2001) Mitochondrial DNA mutations in human disease. Am. J. Med. Genet. 106, 18–26.

    Article  PubMed  CAS  Google Scholar 

  28. Wallace, D. C., Shoffner, J. M., Trounce, I., et al. (1995) Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim. Biophys. Acta 1271, 141–151.

    PubMed  Google Scholar 

  29. Bowling, A. C. and Beal, M. F. (1995) Bioenergetic and oxidative stress in neurodegenerative diseases. Life Sci. 56, 1151–1171.

    Article  PubMed  CAS  Google Scholar 

  30. Schapira, A. H. V. (1998) Mitochondrial dysfunction in neurodegenerative disorders. Biochim. Biophys. Acta 1366, 225–233.

    Article  PubMed  CAS  Google Scholar 

  31. Wallace, D. C. (1994) Mitochondrial DNA mutations in diseases of energy metabolism. J. Bioenerg. Biomembr. 26, 241–250.

    Article  PubMed  CAS  Google Scholar 

  32. Penta, J. S., Johnson, F. H., Wachsman, J. T., and Copeland, W. C. (2001) Mitochondrial DNA in human malignancy. Mutat. Res. 488, 119–133.

    Article  PubMed  CAS  Google Scholar 

  33. Hudson, E. X., Hogue, B. A., Souza-Pinto, N. C., et al. (1998) Age-associated change in mitochondrial DNA damage. Free Radic. Res. 29, 573–579.

    Article  PubMed  CAS  Google Scholar 

  34. Cadenas E. and Davies, K. J. (2000) Mitochondrial free radical generation, oxidative stress, and aging. Free Radic. Biol. Med. 29, 222–230.

    Article  PubMed  CAS  Google Scholar 

  35. Mandavilli, B. S., Santos, J. H., and Van Houten, B. (2002) Mitochondrial DNA repair and aging. Mutat. Res. 509, 127–151.

    PubMed  CAS  Google Scholar 

  36. Boveris, A. and Cadenas, E. (1982) Superoxide and hydrogen peroxide in mitochondria, in Free Radicals in Biology, (Pryor, W. A., ed.). Academic Press, San Diego, CA, pp. 65–90.

    Google Scholar 

  37. Turrens, J. F. and Boveris, A. (1980) Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191, 421–427.

    PubMed  CAS  Google Scholar 

  38. Beckman, K. B. and Ames, B. N. (1999) Endogenous oxidative damage of mtDNA. Mutat. Res. 424, 51–58.

    PubMed  CAS  Google Scholar 

  39. Kowaltowski, A. J. and Vercesi, A. E. (1999) Mitochondrial damage induced by conditions of oxidative stress. Free Radic. Biol. Med. 26, 463–471.

    Article  PubMed  CAS  Google Scholar 

  40. Sawyer, D. E. and Van Houten, B. (1999) Repair of DNA damage in mitochondria. Mutat. Res. 434, 161–176.

    PubMed  CAS  Google Scholar 

  41. Massa, E. M. and Giulivi, C. (1993) Alkoxyl and methyl radical formation during cleavage of tert-butyl hydroperoxide by a mitochondrial membrane-bound, redox active copper pool: an EPR study. Free Radic. Biol. Med. 14, 559–565.

    Article  PubMed  CAS  Google Scholar 

  42. Walter, P. B., Beckman, K. B., and Ames, B.N. (1999) The role of iron and mitochondria in aging, in Understanding the Process of Aging: The Roles of Mitochondria, Free Radicals, and Antioxidants (Cadenas, E., and Packers, L., eds.). Marcel Dekker, New York, NY, pp. 203–227.

    Google Scholar 

  43. Croteau, D. L., Stierum, R. H., and Bohr, V. A. (1999) Mitochondrial DNA repair pathways. Mutat. Res. 434, 137–148.

    PubMed  CAS  Google Scholar 

  44. Bohr, V. A. (2002) Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic. Biol. Med. 32, 804–812.

    Article  PubMed  CAS  Google Scholar 

  45. Zastawny, T. H., Dabrowska, M., Jaskolski, T., et al. (1998) Comparison of oxidative base damage in mitochondrial and nuclear DNA. Free Radic. Biol. Med. 24, 722–725.

    Article  PubMed  CAS  Google Scholar 

  46. Richter, C., Park, J. W., and Ames, B. N. (1998) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. USA 85, 6465–6467.

    Article  Google Scholar 

  47. Mecocci, P., MacGarvey, U., Kaufman, A.E., et al. (1993) Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain. Ann. Neurol. 34, 609–616.

    Article  PubMed  CAS  Google Scholar 

  48. Mecocci, P., MacGarvey, U., and Beal, M. F. (1994) Oxidative damage to mitochondrial DNA is increased in Alzheimer’s disease. Ann. Neurol. 36, 747–751.

    Article  PubMed  CAS  Google Scholar 

  49. Helbock, H. J., Beckman, K. B., Shigenaga, M. K., et al. (1998) DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxoguanine. Proc. Natl. Acad. Sci. USA 95, 288–293.

    Article  PubMed  CAS  Google Scholar 

  50. Anson, R. M., Hudson, E., and Bohr, V. A. (2000) Mitochondrial endogenous oxidative damage has been overestimated. FASEB J. 14, 355–360.

    PubMed  CAS  Google Scholar 

  51. Termini, J. (2000) Hydroperoxide-induced DNA damage and mutations. Mutat. Res. 450, 107–124.

    PubMed  CAS  Google Scholar 

  52. Quan, T. and States, J. C. (1996) Preferential DNA damage in the p53 gene by benzo[a]pyrene metabolites in cytochrome P4501A1-expressing xeroderma pigmentosum group A cells. Mol. Carcinogen. 16, 32–43.

    Article  CAS  Google Scholar 

  53. Cheng, S., Chen, Y., Monforte, J. A., Higuchi, R., and Van Houten, B. (1995) Template integrity is essential for PCR amplification of 20-to 30-kb sequences from genomic DNA. PCR Methods Appl. 4, 294–298.

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

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

    Janine H. Santos, Joel N. Meyer, Bhaskar S. Mandavilli & Bennett Van Houten

Authors
  1. Janine H. Santos
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  2. Joel N. Meyer
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  3. Bhaskar S. Mandavilli
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  4. Bennett Van Houten
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Editors and Affiliations

  1. Pharmacological Sciences, SUNY Stony Brook, Stony Brook, NY

    Daryl S. Henderson

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© 2006 Humama Press Inc.

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Santos, J.H., Meyer, J.N., Mandavilli, B.S., Van Houten, B. (2006). Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:183

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  • DOI: https://doi.org/10.1385/1-59259-973-7:183

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-513-2

  • Online ISBN: 978-1-59259-973-8

  • eBook Packages: Springer Protocols

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