Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Sep;11(9):4537–4544. doi: 10.1128/mcb.11.9.4537

Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation.

D Ramotar 1, S C Popoff 1, E B Gralla 1, B Demple 1
PMCID: PMC361329  PMID: 1715020

Abstract

The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.

Full text

PDF
4537

Selected References

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

  1. Barrows L. R., Magee P. N. Nonenzymatic methylation of DNA by S-adenosylmethionine in vitro. Carcinogenesis. 1982;3(3):349–351. doi: 10.1093/carcin/3.3.349. [DOI] [PubMed] [Google Scholar]
  2. Cunningham R. P., Saporito S. M., Spitzer S. G., Weiss B. Endonuclease IV (nfo) mutant of Escherichia coli. J Bacteriol. 1986 Dec;168(3):1120–1127. doi: 10.1128/jb.168.3.1120-1127.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Demple B., Johnson A., Fung D. Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7731–7735. doi: 10.1073/pnas.83.20.7731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Friedberg E. C. Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae. Microbiol Rev. 1988 Mar;52(1):70–102. doi: 10.1128/mr.52.1.70-102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Giloni L., Takeshita M., Johnson F., Iden C., Grollman A. P. Bleomycin-induced strand-scission of DNA. Mechanism of deoxyribose cleavage. J Biol Chem. 1981 Aug 25;256(16):8608–8615. [PubMed] [Google Scholar]
  6. Greenberg J. T., Demple B. Overproduction of peroxide-scavenging enzymes in Escherichia coli suppresses spontaneous mutagenesis and sensitivity to redox-cycling agents in oxyR-mutants. EMBO J. 1988 Aug;7(8):2611–2617. doi: 10.1002/j.1460-2075.1988.tb03111.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Henner W. D., Grunberg S. M., Haseltine W. A. Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks. J Biol Chem. 1983 Dec 25;258(24):15198–15205. [PubMed] [Google Scholar]
  8. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  9. Hutchinson F. Chemical changes induced in DNA by ionizing radiation. Prog Nucleic Acid Res Mol Biol. 1985;32:115–154. doi: 10.1016/s0079-6603(08)60347-5. [DOI] [PubMed] [Google Scholar]
  10. Imlay J. A., Linn S. DNA damage and oxygen radical toxicity. Science. 1988 Jun 3;240(4857):1302–1309. doi: 10.1126/science.3287616. [DOI] [PubMed] [Google Scholar]
  11. Johnson A. W., Demple B. Yeast DNA 3'-repair diesterase is the major cellular apurinic/apyrimidinic endonuclease: substrate specificity and kinetics. J Biol Chem. 1988 Dec 5;263(34):18017–18022. [PubMed] [Google Scholar]
  12. Johnson A. W., Demple B. Yeast DNA diesterase for 3'-fragments of deoxyribose: purification and physical properties of a repair enzyme for oxidative DNA damage. J Biol Chem. 1988 Dec 5;263(34):18009–18016. [PubMed] [Google Scholar]
  13. Levin J. D., Demple B. Analysis of class II (hydrolytic) and class I (beta-lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate. Nucleic Acids Res. 1990 Sep 11;18(17):5069–5075. doi: 10.1093/nar/18.17.5069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Levin J. D., Johnson A. W., Demple B. Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. J Biol Chem. 1988 Jun 15;263(17):8066–8071. [PubMed] [Google Scholar]
  15. Lindahl T., Andersson A. Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3618–3623. doi: 10.1021/bi00769a019. [DOI] [PubMed] [Google Scholar]
  16. Lindahl T. DNA repair enzymes. Annu Rev Biochem. 1982;51:61–87. doi: 10.1146/annurev.bi.51.070182.000425. [DOI] [PubMed] [Google Scholar]
  17. Lindahl T., Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3610–3618. doi: 10.1021/bi00769a018. [DOI] [PubMed] [Google Scholar]
  18. Lindahl T., Sedgwick B., Sekiguchi M., Nakabeppu Y. Regulation and expression of the adaptive response to alkylating agents. Annu Rev Biochem. 1988;57:133–157. doi: 10.1146/annurev.bi.57.070188.001025. [DOI] [PubMed] [Google Scholar]
  19. Loeb L. A., Preston B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet. 1986;20:201–230. doi: 10.1146/annurev.ge.20.120186.001221. [DOI] [PubMed] [Google Scholar]
  20. Moore C. W. Isolation and partial characterization of mutants of Saccharomyces cerevisiae altered in sensitivities to lethal effects of bleomycins. J Antibiot (Tokyo) 1980 Nov;33(11):1369–1375. doi: 10.7164/antibiotics.33.1369. [DOI] [PubMed] [Google Scholar]
  21. Pierce M. K., Giroux C. N., Kunz B. A. Development of a yeast system to assay mutational specificity. Mutat Res. 1987 Apr;182(2):65–74. doi: 10.1016/0165-1161(87)90055-0. [DOI] [PubMed] [Google Scholar]
  22. Popoff S. C., Spira A. I., Johnson A. W., Demple B. Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4193–4197. doi: 10.1073/pnas.87.11.4193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Povirk L. F., Goldberg I. H. Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis. Proc Natl Acad Sci U S A. 1985 May;82(10):3182–3186. doi: 10.1073/pnas.82.10.3182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prakash L., Prakash S. Isolation and characterization of MMS-sensitive mutants of Saccharomyces cerevisiae. Genetics. 1977 May;86(1):33–55. doi: 10.1093/genetics/86.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reynolds R. J. Induction and repair of closely opposed pyrimidine dimers in Saccharomyces cerevisiae. Mutat Res. 1987 Nov;184(3):197–207. doi: 10.1016/0167-8817(87)90017-4. [DOI] [PubMed] [Google Scholar]
  26. Rose M. D. Isolation of genes by complementation in yeast. Methods Enzymol. 1987;152:481–504. doi: 10.1016/0076-6879(87)52056-0. [DOI] [PubMed] [Google Scholar]
  27. Rydberg B., Lindahl T. Nonenzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-L-methionine is a potentially mutagenic reaction. EMBO J. 1982;1(2):211–216. doi: 10.1002/j.1460-2075.1982.tb01149.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shigenaga M. K., Gimeno C. J., Ames B. N. Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9697–9701. doi: 10.1073/pnas.86.24.9697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Struhl K., Davis R. W. A physical, genetic and transcriptional map of the cloned his3 gene region of Saccharomyces cerevisiae. J Mol Biol. 1980 Jan 25;136(3):309–332. doi: 10.1016/0022-2836(80)90376-9. [DOI] [PubMed] [Google Scholar]
  30. Von Borstel R. C. Measuring spontaneous mutation rates in yeast. Methods Cell Biol. 1978;20:1–24. doi: 10.1016/s0091-679x(08)62005-1. [DOI] [PubMed] [Google Scholar]
  31. Walker G. C. Inducible DNA repair systems. Annu Rev Biochem. 1985;54:425–457. doi: 10.1146/annurev.bi.54.070185.002233. [DOI] [PubMed] [Google Scholar]
  32. Wallace S. S. AP endonucleases and DNA glycosylases that recognize oxidative DNA damage. Environ Mol Mutagen. 1988;12(4):431–477. doi: 10.1002/em.2860120411. [DOI] [PubMed] [Google Scholar]
  33. Weiss B., Grossman L. Phosphodiesterases involved in DNA repair. Adv Enzymol Relat Areas Mol Biol. 1987;60:1–34. doi: 10.1002/9780470123065.ch1. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES