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

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Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1105))

Abstract

In this chapter, we describe a gene-specific quantitative PCR (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been used extensively to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins and has proven to be particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA damage. QPCR can be used to quantify both 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 mitochondrial DNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol of the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.

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References

  1. Ponti M, Forrow SM, Souhami RL, D'Incalci M, Hartley JA (1991) Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase. Nucleic Acids Res 19:2929–2933

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Kalinowski DP, Illenye S, 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  CAS  PubMed Central  PubMed  Google Scholar 

  4. Van Houten B, Cheng S, 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

    Article  PubMed  Google Scholar 

  5. Van Houten B, Chen Y, Nicklas JA, Rainville IR, O'Neill JP (1998) Development of long PCR techniques to analyze deletion mutations of the human hprt gene. Mutat Res 403: 171–175

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Yakes FM, 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 U S A 94:514–519

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Moon SK, Thompson LJ, Madamanchi N et al (2001) Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 280:H2779–H2788

    CAS  PubMed  Google Scholar 

  10. Denissenko MF, Cahill J, Koudriakova TB, Gerber N, Pfeifer GP (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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Jin GF, Hurst JS, Godley BF (2001) Rod outer segments mediate mitochondrial DNA damage and apoptosis in human retinal pigment epithelium. Curr Eye Res 23:11–19

    Article  CAS  PubMed  Google Scholar 

  13. Sawyer DE, Mercer BG, Wiklendt AM, Aitken RJ (2003) Quantitative analysis of gene-specific DNA damage in human spermatozoa. Mutat Res 529:21–34

    Article  CAS  PubMed  Google Scholar 

  14. Santos JH, Hunakova L, Chen Y, Bortner C, 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  CAS  PubMed  Google Scholar 

  15. Yanez JA, Teng XW, Roupe KA, Fariss MW, Davies NM (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, Patierno SR (2001) Effects of glutathione on chromium-induced DNA crosslinking and DNA polymerase arrest. Mol Cell Biochem 222:173–182

    Article  PubMed  Google Scholar 

  17. Chandrasekhar D, Van Houten B (1994) High resolution mapping of UV-induced photoproducts in the Escherichia coli lacI gene: Inefficient repair of the non-transcribed strand correlates with high mutation frequency. J Mol Biol 238:319–332

    Article  CAS  PubMed  Google Scholar 

  18. Yakes FM, Chen Y, Van Houten B (1996) PCR-based assays for the detection and quantitation of DNA damage and repair. In: Pfeifer GP (ed) Technologies for detection of DNA damage and mutations. Plenum Press, New York, NY, pp 171–184

    Chapter  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  20. Chen KH, Yakes FM, Srivastava DK 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  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. Deng G, Su JH, Ivins KJ, Van Houten B, Cotman CW (1999) Bcl-2 facilitates recovery from DNA damage after oxidative stress. Exptl Neurol 159:309–318

    Article  CAS  Google Scholar 

  23. Ballinger SW, Patterson C, Yan CN et al (2000) Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res 86:960–966

    Article  CAS  PubMed  Google Scholar 

  24. Chandrasekhar D, 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 Escherichia coli. Mutat Res 450:19–40

    Article  CAS  PubMed  Google Scholar 

  25. Sobol RW, Watson DE, Nakamura J et al (2002) Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress. Proc Natl Acad Sci U S A 99:6860–6865

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Jung D, Cho Y, Collins LB, Swenberg JA, Di Giulio RT (2009) Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site. Aquat Toxicol 95:44–51

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Trnka J, Blaikie FH, Logan A, Smith RA, Murphy MP (2009) Antioxidant properties of MitoTEMPOL and its hydroxylamine. Free Radic Res 43:4–12

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Lu B, Yadav S, Shah PG et al. Roles for the human ATP-dependent Lon protease in mitochondrial DNA maintenance. J Biol Chem 282:17363–17374

    Google Scholar 

  29. Ahmed S, Passos JF, Birket MJ et al (2008) Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress. J Cell Sci 121: 1046–1053

    Article  CAS  PubMed  Google Scholar 

  30. Rothfuss O, Fischer H, Hasegawa T et al (2009) Parkin protects mitochondrial genome integrity and supports mitochondrial DNA repair. Hum Mol Genet 18:3832–3850

    Article  CAS  PubMed  Google Scholar 

  31. Chatterjee A, Mambo E, Zhang Y, Deweese T, Sidransky D (2006) Targeting of mutant hogg1 in mammalian mitochondria and nucleus: effect on cellular survival upon oxidative stress. BMC Cancer 6:235

    Article  PubMed Central  PubMed  Google Scholar 

  32. Maloney SC, Adair JE, Smerdon MJ, Reeves R (2007) Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins. DNA Repair 6:1371–1379

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Boyd WA, Crocker TL, Rodriguez AM et al (2010) Nucleotide excision repair genes are expressed at low levels and are not detectably inducible in Caenorhabditis elegans somatic tissues, but their function is required for normal adult life after UVC exposure. Mutat Res 683:57–67

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Ma W, Panduri V, Sterling JF, Van Houten B, Gordenin DA, Resnick MA (2009) The transition of closely opposed lesions to double-strand breaks during long-patch base excision repair is prevented by the coordinated action of DNA polymerase ∂ and Rad27/Fen1. Mol Cell Biol 29:1212–1221

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Liu P, Qian L, Sung JS et al (2008) Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria. Mol Cell Biol 28:4975–4987

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Salmon AB, Ljungman M, Miller RA (2008) Cells from long-lived mutant mice exhibit enhanced repair of ultraviolet lesions. J Gerontol A Biol Sci Med Sci 63:219–231

    Article  PubMed Central  PubMed  Google Scholar 

  37. Acevedo-Torres K, Berrios L, Rosario N et al (2009) Mitochondrial DNA damage is a hallmark of chemically induced and the R6/2 transgenic model of Huntington’s disease. DNA Repair 8:126–136

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Mao L, Wertzler KJ, Maloney SC, Wang Z, Magnuson NS, Reeves R (2009) HMGA1 levels influence mitochondrial function and mitochondrial DNA repair efficiency. Mol Cell Biol 29:5426–5440

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Jung D, Cho Y, Meyer JN, Di Giulio RT (2009) The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus). Comp Biochem Physiol C Toxicol Pharmacol 149:182–186

    Article  PubMed Central  PubMed  Google Scholar 

  40. Duxin JP, Dao B, Martinsson P et al (2009) Human Dna2 is a nuclear and mitochondrial DNA maintenance protein. Mol Cell Biol 29:4274–4282

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Acevedo-Torres K, Fonseca-Williams S, Ayala-Torres S, Torres-Ramos CA (2009) Requirement of the Saccharomyces cerevisiae APN1 gene for the repair of mitochondrial DNA alkylation damage. Environ Mol Mutagen 50:317–327

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Wang AL, Lukas TJ, Yuan M, Du N, Tso MO, Neufeld AH (2009) Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration. PLoS One 4:e4160

    Article  PubMed Central  PubMed  Google Scholar 

  43. Wang AL, Lukas TJ, Yuan M, Neufeld AH (2008) Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid. Mol Vis 14:644–651

    PubMed Central  PubMed  Google Scholar 

  44. Bonner M, Kmiec EB (2009) DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides. Mutat Res 669:85–94

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Khurana RN, Parikh JG, Saraswathy S, Wu GS, Rao NA (2008) Mitochondrial oxidative DNA damage in experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 49:3299–3304

    Article  PubMed  Google Scholar 

  46. Haugen AC, Di Prospero NA, Parker JS et al (2010) Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: cellular model of pathology. PLoS Genet 6:e1000812

    Article  PubMed Central  PubMed  Google Scholar 

  47. Meyer JN, Boyd WA, Azzam GA, Haugen AC, Freedman JH, Van Houten B (2007) Decline of nucleotide excision repair capacity in aging Caenorhabditis elegans. Genome Biol 8:R70

    Article  PubMed Central  PubMed  Google Scholar 

  48. Meyer JN (2010) QPCR: a tool for analysis of mitochondrial and nuclear DNA damage in ecotoxicology. Ecotoxicology 19:804–811

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Hunter SE, Jung D, Di Giulio RT, Meyer JN (2010) The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number. Methods 51:444–451

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Kovalenko OA, Santos JH (2009) Analysis of oxidative damage by gene-specific quantitative PCR. Curr Protoc Hum Genet 62:19.1.1– 19.1.13

    Google Scholar 

  51. Song GJ, Lewis V (2008) Mitochondrial DNA integrity and copy number in sperm from infertile men. Fertil Steril 90:2238–2244

    Article  CAS  PubMed  Google Scholar 

  52. Edwards JG (2009) Quantification of mitochondrial DNA (mtDNA) damage and error rates by real-time QPCR. Mitochondrion 9: 31–35

    Article  CAS  PubMed  Google Scholar 

  53. Wallace DC (1999) Mitochondrial diseases in man and mouse. Science 283:1482–1488

    Article  CAS  PubMed  Google Scholar 

  54. DiMauro S, Schon EA (2001) Mitochondrial DNA mutations in human disease. Am J Med Genet 106:18–26

    Article  CAS  PubMed  Google Scholar 

  55. Wallace DC, Shoffner JM, Trounce I et al (1995) Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim Biophys Acta 1271:141–151

    Article  PubMed  Google Scholar 

  56. Bowling AC, Beal MF (1995) Bioenergetic and oxidative stress in neurodegenerative diseases. Life Sci 56:1151–1171

    Article  CAS  PubMed  Google Scholar 

  57. Schapira AH (1998) Mitochondrial dysfunction in neurodegenerative disorders. Biochim Biophys Acta 1366:225–233

    Article  CAS  PubMed  Google Scholar 

  58. Wallace DC (1994) Mitochondrial DNA mutations in diseases of energy metabolism. J Bioenerg Biomembr 26:241–250

    Article  CAS  PubMed  Google Scholar 

  59. Penta JS, Johnson FM, Wachsman JT, Copeland WC (2001) Mitochondrial DNA in human malignancy. Mutat Res 488: 119–133

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  61. Hudson EK, Hogue BA, Souza-Pinto NC et al (1998) Age-associated change in mitochondrial DNA damage. Free Radic Res 29: 573–579

    Article  CAS  PubMed  Google Scholar 

  62. Mandavilli BS, Santos JH, Van Houten B (2002) Mitochondrial DNA repair and aging. Mutat Res 509:127–151

    Article  CAS  PubMed  Google Scholar 

  63. Boveris A, Cadenas E (1982) Superoxide and hydrogen peroxide in mitochondria. In: Pryor WA (ed) Free radicals in biology. Academic, San Diego, CA, pp 65–90

    Google Scholar 

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

    CAS  PubMed Central  PubMed  Google Scholar 

  65. Beckman KB, Ames BN (1999) Endogenous oxidative damage of mtDNA. Mutat Res 424: 51–58

    Article  CAS  PubMed  Google Scholar 

  66. Kowaltowski AJ, Vercesi AE (1999) Mitochondrial damage induced by conditions of oxidative stress. Free Radic Biol Med 26: 463–471

    Article  CAS  PubMed  Google Scholar 

  67. Sawyer DE, Van Houten B (1999) Repair of DNA damage in mitochondria. Mutat Res 434:161–176

    Article  CAS  PubMed  Google Scholar 

  68. Massa EM, 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  CAS  PubMed  Google Scholar 

  69. Walter PB, Beckman KB, Ames BN (1999) The role of iron and mitochondria in aging. In: Cadenas E, Packers L (eds) Understanding the process of aging: the roles of mitochondria, free radicals, and antioxidants. Marcel Dekker, New York, NY, pp 203–227

    Google Scholar 

  70. Croteau DL, Stierum RH, Bohr VA (1999) Mitochondrial DNA repair pathways. Mutat Res 434:137–148

    Article  CAS  PubMed  Google Scholar 

  71. 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 32:804–812

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  73. Mecocci P, MacGarvey U, Kaufman AE, Koontz D, Shoffner JM, Wallace DC et al (1993) Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain. Ann Neurol 34(4):609–616

    Article  CAS  PubMed  Google Scholar 

  74. Zastawny TH, 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  CAS  PubMed  Google Scholar 

  75. Helbock HJ, Beckman KB, Shigenaga MK et al (1998) DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc Natl Acad Sci U S A 95:288–293

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  76. Anson RM, Hudson E, Bohr VA (2000) Mitochondrial endogenous oxidative damage has been overestimated. FASEB J 14:355–360

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  79. Lu T, Pan Y, Kao SY et al (2004) Gene regulation and DNA damage in the ageing human brain. Nature 429:883–891

    Article  CAS  PubMed  Google Scholar 

  80. Cheng S, Chen Y, Monforte JA, Higuchi R, 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

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA

    Amy Furda, Janine H. Santos, Joel N. Meyer & Bennett Van Houten

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  1. Amy Furda
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  2. Janine H. Santos
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  3. Joel N. Meyer
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  4. Bennett Van Houten
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Correspondence to Bennett Van Houten .

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Editors and Affiliations

  1. Occupational Health, University of Pittsburgh Dept. Environmental &, Pittsburgh, Pennsylvania, USA

    Phouthone Keohavong

  2. Dept. of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, Florida, USA

    Stephen G. Grant

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Furda, A., Santos, J.H., Meyer, J.N., Van Houten, B. (2014). Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_31

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  • DOI: https://doi.org/10.1007/978-1-62703-739-6_31

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