Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Nov 1;25(21):4362–4369. doi: 10.1093/nar/25.21.4362

Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA.

W J Driggers 1, G P Holmquist 1, S P LeDoux 1, G L Wilson 1
PMCID: PMC147038  PMID: 9336469

Abstract

Genomic DNA is constantly being damaged and repaired and our genomes exist at lesion equilibrium for damage created by endogenous mutagens. Mitochondrial DNA (mtDNA) has the highest lesion equilibrium frequency recorded; presumably due to damage by H2O2 and free radicals generated during oxidative phosphorylation processes. We measured the frequencies of single strand breaks and oxidative base damage in mtDNA by ligation-mediated PCR and a quantitative Southern blot technique coupled with digestion by the enzymes endonuclease III and formamidopyrimidine DNA glycosylase. Addition of 5 mM alloxan to cultured rat cells increased the rate of oxidative base damage and, by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the single strand breaks and oxidative base damage frequency decreased to levels slightly below normal at 4 h and returned to normal levels at 8 h, the overshoot at 4 h being attributed to an adaptive up-regulation of mitochondrial excision repair activity. Guanine positions showed the highest endogenous lesion frequencies and were the most responsive positions to alloxan-induced oxidative stress. Although specific bases were consistently hot spots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner. The data reveal non-random oxidative damage to several nucleotides in mtDNA and an apparent adaptive, non-strand selective response for removal of such damage. These are the first studies to characterize oxidative damage and its subsequent removal at the nucleotide level in mtDNA.

Full Text

The Full Text of this article is available as a PDF (321.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ames B. N. Endogenous DNA damage as related to cancer and aging. Mutat Res. 1989 Sep;214(1):41–46. doi: 10.1016/0027-5107(89)90196-6. [DOI] [PubMed] [Google Scholar]
  2. Ames B. N., Shigenaga M. K., Hagen T. M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7915–7922. doi: 10.1073/pnas.90.17.7915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson S., Bankier A. T., Barrell B. G., de Bruijn M. H., Coulson A. R., Drouin J., Eperon I. C., Nierlich D. P., Roe B. A., Sanger F. Sequence and organization of the human mitochondrial genome. Nature. 1981 Apr 9;290(5806):457–465. doi: 10.1038/290457a0. [DOI] [PubMed] [Google Scholar]
  4. Barres B. A., Hart I. K., Coles H. S., Burne J. F., Voyvodic J. T., Richardson W. D., Raff M. C. Cell death and control of cell survival in the oligodendrocyte lineage. Cell. 1992 Jul 10;70(1):31–46. doi: 10.1016/0092-8674(92)90531-g. [DOI] [PubMed] [Google Scholar]
  5. Cadenas E. Biochemistry of oxygen toxicity. Annu Rev Biochem. 1989;58:79–110. doi: 10.1146/annurev.bi.58.070189.000455. [DOI] [PubMed] [Google Scholar]
  6. Chance B., Sies H., Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev. 1979 Jul;59(3):527–605. doi: 10.1152/physrev.1979.59.3.527. [DOI] [PubMed] [Google Scholar]
  7. Clayton D. A. Replication and transcription of vertebrate mitochondrial DNA. Annu Rev Cell Biol. 1991;7:453–478. doi: 10.1146/annurev.cb.07.110191.002321. [DOI] [PubMed] [Google Scholar]
  8. Clayton D. A. Transcription of the mammalian mitochondrial genome. Annu Rev Biochem. 1984;53:573–594. doi: 10.1146/annurev.bi.53.070184.003041. [DOI] [PubMed] [Google Scholar]
  9. Cortopassi G. A., Arnheim N. Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic Acids Res. 1990 Dec 11;18(23):6927–6933. doi: 10.1093/nar/18.23.6927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dizdaroglu M., Laval J., Boiteux S. Substrate specificity of the Escherichia coli endonuclease III: excision of thymine- and cytosine-derived lesions in DNA produced by radiation-generated free radicals. Biochemistry. 1993 Nov 16;32(45):12105–12111. doi: 10.1021/bi00096a022. [DOI] [PubMed] [Google Scholar]
  11. Driggers W. J., LeDoux S. P., Wilson G. L. Repair of oxidative damage within the mitochondrial DNA of RINr 38 cells. J Biol Chem. 1993 Oct 15;268(29):22042–22045. [PubMed] [Google Scholar]
  12. Frenkel K. Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacol Ther. 1992;53(1):127–166. doi: 10.1016/0163-7258(92)90047-4. [DOI] [PubMed] [Google Scholar]
  13. Gao S., Drouin R., Holmquist G. P. DNA repair rates mapped along the human PGK1 gene at nucleotide resolution. Science. 1994 Mar 11;263(5152):1438–1440. doi: 10.1126/science.8128226. [DOI] [PubMed] [Google Scholar]
  14. Gazdar A. F., Chick W. L., Oie H. K., Sims H. L., King D. L., Weir G. C., Lauris V. Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3519–3523. doi: 10.1073/pnas.77.6.3519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jackson D. A., Bartlett J., Cook P. R. Sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in human cells: the role of transcription units. Nucleic Acids Res. 1996 Apr 1;24(7):1212–1219. doi: 10.1093/nar/24.7.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jaruga P., Dizdaroglu M. Repair of products of oxidative DNA base damage in human cells. Nucleic Acids Res. 1996 Apr 15;24(8):1389–1394. doi: 10.1093/nar/24.8.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim S. T., Malhotra K., Smith C. A., Taylor J. S., Sancar A. DNA photolyase repairs the trans-syn cyclobutane thymine dimer. Biochemistry. 1993 Jul 20;32(28):7065–7068. doi: 10.1021/bi00079a001. [DOI] [PubMed] [Google Scholar]
  18. LeDoux S. P., Patton N. J., Avery L. J., Wilson G. L. Repair of N-methylpurines in the mitochondrial DNA of xeroderma pigmentosum complementation group D cells. Carcinogenesis. 1993 May;14(5):913–917. doi: 10.1093/carcin/14.5.913. [DOI] [PubMed] [Google Scholar]
  19. LeDoux S. P., Wilson G. L., Beecham E. J., Stevnsner T., Wassermann K., Bohr V. A. Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells. Carcinogenesis. 1992 Nov;13(11):1967–1973. doi: 10.1093/carcin/13.11.1967. [DOI] [PubMed] [Google Scholar]
  20. Levy D. D., Magee A. D., Seidman M. M. Single nucleotide positions have proximal and distal influence on UV mutation hotspots and coldspots. J Mol Biol. 1996 May 3;258(2):251–260. doi: 10.1006/jmbi.1996.0247. [DOI] [PubMed] [Google Scholar]
  21. Pettepher C. C., LeDoux S. P., Bohr V. A., Wilson G. L. Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin. J Biol Chem. 1991 Feb 15;266(5):3113–3117. [PubMed] [Google Scholar]
  22. Pfeifer G. P., Drouin R., Holmquist G. P. Detection of DNA adducts at the DNA sequence level by ligation-mediated PCR. Mutat Res. 1993 Jul;288(1):39–46. doi: 10.1016/0027-5107(93)90206-u. [DOI] [PubMed] [Google Scholar]
  23. Pfeifer G. P., Drouin R., Riggs A. D., Holmquist G. P. In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1374–1378. doi: 10.1073/pnas.88.4.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pikó L. Accumulation of mtDNA defects and changes in mtDNA content in mouse and rat tissues with aging. Ann N Y Acad Sci. 1992 Nov 21;663:450–452. doi: 10.1111/j.1749-6632.1992.tb38698.x. [DOI] [PubMed] [Google Scholar]
  25. Poulton J., Deadman M. E., Bindoff L., Morten K., Land J., Brown G. Families of mtDNA re-arrangements can be detected in patients with mtDNA deletions: duplications may be a transient intermediate form. Hum Mol Genet. 1993 Jan;2(1):23–30. doi: 10.1093/hmg/2.1.23. [DOI] [PubMed] [Google Scholar]
  26. Richter C. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol. 1995 Jul;27(7):647–653. doi: 10.1016/1357-2725(95)00025-k. [DOI] [PubMed] [Google Scholar]
  27. Richter C., Park J. W., Ames B. N. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6465–6467. doi: 10.1073/pnas.85.17.6465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Richter C. Reactive oxygen and DNA damage in mitochondria. Mutat Res. 1992 Sep;275(3-6):249–255. doi: 10.1016/0921-8734(92)90029-o. [DOI] [PubMed] [Google Scholar]
  29. Rodriguez H., Drouin R., Holmquist G. P., O'Connor T. R., Boiteux S., Laval J., Doroshow J. H., Akman S. A. Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J Biol Chem. 1995 Jul 21;270(29):17633–17640. doi: 10.1074/jbc.270.29.17633. [DOI] [PubMed] [Google Scholar]
  30. Ruven H. J., Seelen C. M., Lohman P. H., van Kranen H., van Zeeland A. A., Mullenders L. H. Strand-specific removal of cyclobutane pyrimidine dimers from the p53 gene in the epidermis of UVB-irradiated hairless mice. Oncogene. 1994 Dec;9(12):3427–3432. [PubMed] [Google Scholar]
  31. Sancar A., Sancar G. B. DNA repair enzymes. Annu Rev Biochem. 1988;57:29–67. doi: 10.1146/annurev.bi.57.070188.000333. [DOI] [PubMed] [Google Scholar]
  32. Soong N. W., Hinton D. R., Cortopassi G., Arnheim N. Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain. Nat Genet. 1992 Dec;2(4):318–323. doi: 10.1038/ng1292-318. [DOI] [PubMed] [Google Scholar]
  33. Tchou J., Bodepudi V., Shibutani S., Antoshechkin I., Miller J., Grollman A. P., Johnson F. Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA. J Biol Chem. 1994 May 27;269(21):15318–15324. [PubMed] [Google Scholar]
  34. Wallace S. S. DNA damages processed by base excision repair: biological consequences. Int J Radiat Biol. 1994 Nov;66(5):579–589. doi: 10.1080/09553009414551661. [DOI] [PubMed] [Google Scholar]
  35. Zhang C., Baumer A., Maxwell R. J., Linnane A. W., Nagley P. Multiple mitochondrial DNA deletions in an elderly human individual. FEBS Lett. 1992 Feb 3;297(1-2):34–38. doi: 10.1016/0014-5793(92)80321-7. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES