Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Oct 15;24(20):4057–4062. doi: 10.1093/nar/24.20.4057

Repair of DNA damage in a mitochondrial lysate of Xenopus laevis oocytes.

M Ryoji 1, H Katayama 1, H Fusamae 1, A Matsuda 1, F Sakai 1, H Utano 1
PMCID: PMC146209  PMID: 8918812

Abstract

We examined DNA repair activities of a mitochondrial lysate derived from Xenopus laevis oocytes. Plasmid DNA, exposed to HCl, H2O2 or UV light, was used as the substrate for the in vitro repair reaction. DNA synthesis in the lysate was stimulated 2-8-fold by such lesions, indicating the presence of excision repair activities. This repair DNA synthesis was not affected by aphidicolin, but was sensitive to N-ethylmaleimide. Thus the mitochondrial DNA polymerase, i.e., pol gamma is indeed involved in the reaction. Actual repair of the depurinated DNA was demonstrated by using the polymerase chain reaction (PCR), where the amount of the amplified DNA fragment increased significantly if the depurinated template was incubated in the lysate prior to the PCR. UV-irradiated DNA, on the other hand, restored its ability as a PCR template only if the repair reaction was carried out under the light. Therefore, in this system, UV-induced damage is repaired mainly by photoreactivation. These results show that mitochondria of Xenopus oocytes possess excision repair as well as photolyase activities, and that the in vitro repair system described here should be useful for further molecular characterization of such DNA repair machinery.

Full Text

The Full Text of this article is available as a PDF (102.3 KB).

Selected References

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

  1. Brun G., Vannier P., Scovassi I., Callen J. C. DNA topoisomerase I from mitochondria of Xenopus laevis oocytes. Eur J Biochem. 1981 Aug;118(2):407–415. doi: 10.1111/j.1432-1033.1981.tb06417.x. [DOI] [PubMed] [Google Scholar]
  2. Callen J. C., Tourte M., Dennebouy N., Mounolou J. C. Changes in D-loop frequency and superhelicity among the mitochondrial DNA molecules in relation to organelle biogenesis in oocytes of Xenopus laevis. Exp Cell Res. 1983 Jan;143(1):115–125. doi: 10.1016/0014-4827(83)90114-3. [DOI] [PubMed] [Google Scholar]
  3. Cheng K. C., Cahill D. S., Kasai H., Nishimura S., Loeb L. A. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions. J Biol Chem. 1992 Jan 5;267(1):166–172. [PubMed] [Google Scholar]
  4. Chi N. W., Kolodner R. D. Purification and characterization of MSH1, a yeast mitochondrial protein that binds to DNA mismatches. J Biol Chem. 1994 Nov 25;269(47):29984–29992. [PubMed] [Google Scholar]
  5. Clayton D. A., Doda J. N., Friedberg E. C. The absence of a pyrimidine dimer repair mechanism in mammalian mitochondria. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2777–2781. doi: 10.1073/pnas.71.7.2777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Dunon-Bluteau D., Cordonnier A., Brun G. DNA synthesis in a mitochondrial lysate of Xenopus laevis oocytes. H strand replication in vitro. J Mol Biol. 1987 Sep 20;197(2):175–185. doi: 10.1016/0022-2836(87)90116-1. [DOI] [PubMed] [Google Scholar]
  8. Floyd R. A. Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J. 1990 Jun;4(9):2587–2597. [PubMed] [Google Scholar]
  9. Goscin L. P., Byrnes J. J. DNA polymerase delta: one polypeptide, two activities. Biochemistry. 1982 May 11;21(10):2513–2518. doi: 10.1021/bi00539a034. [DOI] [PubMed] [Google Scholar]
  10. Holt I. J., Harding A. E., Morgan-Hughes J. A. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature. 1988 Feb 25;331(6158):717–719. doi: 10.1038/331717a0. [DOI] [PubMed] [Google Scholar]
  11. Hübscher U., Thömmes P. DNA polymerase epsilon: in search of a function. Trends Biochem Sci. 1992 Feb;17(2):55–58. doi: 10.1016/0968-0004(92)90499-y. [DOI] [PubMed] [Google Scholar]
  12. Ikegami S., Taguchi T., Ohashi M., Oguro M., Nagano H., Mano Y. Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-alpha. Nature. 1978 Oct 5;275(5679):458–460. doi: 10.1038/275458a0. [DOI] [PubMed] [Google Scholar]
  13. Kalinowski D. P., Illenye S., Van Houten B. Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay. Nucleic Acids Res. 1992 Jul 11;20(13):3485–3494. doi: 10.1093/nar/20.13.3485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Krokan H., Schaffer P., DePamphilis M. L. Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid. Biochemistry. 1979 Oct 2;18(20):4431–4443. doi: 10.1021/bi00587a025. [DOI] [PubMed] [Google Scholar]
  15. Lee M. Y., Tan C. K., Downey K. M., So A. G. Structural and functional properties of calf thymus DNA polymerase delta. Prog Nucleic Acid Res Mol Biol. 1981;26:83–96. doi: 10.1016/s0079-6603(08)60396-7. [DOI] [PubMed] [Google Scholar]
  16. Marinos E., Billett F. S. Mitochondrial number, cytochrome oxidase and succinic dehydrogenase activity in Xenopus laevis oocytes. J Embryol Exp Morphol. 1981 Apr;62:395–409. [PubMed] [Google Scholar]
  17. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  18. Myers K. A., Saffhill R., O'Connor P. J. Repair of alkylated purines in the hepatic DNA of mitochondria and nuclei in the rat. Carcinogenesis. 1988 Feb;9(2):285–292. doi: 10.1093/carcin/9.2.285. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Prakash L. Repair of pyrimidine dimers in nuclear and mitochondrial DNA of yeast irradiated with low doses of ultraviolet light. J Mol Biol. 1975 Nov 15;98(4):781–795. doi: 10.1016/s0022-2836(75)80010-6. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Ryoji M., Tominna E., Yasui W. Minichromosome assembly accompanying repair-type DNA synthesis in Xenopus oocytes. Nucleic Acids Res. 1989 Dec 25;17(24):10243–10258. doi: 10.1093/nar/17.24.10243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Satoh M. S., Jones C. J., Wood R. D., Lindahl T. DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6335–6339. doi: 10.1073/pnas.90.13.6335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sugiyama S., Hattori K., Hayakawa M., Ozawa T. Quantitative analysis of age-associated accumulation of mitochondrial DNA with deletion in human hearts. Biochem Biophys Res Commun. 1991 Oct 31;180(2):894–899. doi: 10.1016/s0006-291x(05)81149-0. [DOI] [PubMed] [Google Scholar]
  25. Tomkinson A. E., Bonk R. T., Kim J., Bartfeld N., Linn S. Mammalian mitochondrial endonuclease activities specific for ultraviolet-irradiated DNA. Nucleic Acids Res. 1990 Feb 25;18(4):929–935. doi: 10.1093/nar/18.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tomkinson A. E., Bonk R. T., Linn S. Mitochondrial endonuclease activities specific for apurinic/apyrimidinic sites in DNA from mouse cells. J Biol Chem. 1988 Sep 5;263(25):12532–12537. [PubMed] [Google Scholar]

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

RESOURCES