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. 1981 May 1;195(2):407–417. doi: 10.1042/bj1950407

Apurinic endonuclease from Saccharomyces cerevisiae.

H W Thielmann, U Hess
PMCID: PMC1162904  PMID: 6274306

Abstract

An endonuclease cleaving depurinated and alkylated double-stranded DNA has been purified 500-fold from Saccharomyces cerevisiae, strain MB 1052. The enzyme has an Mr of 31 000 +/- 2000, a sedimentation value of 3.2S and a diffusion coefficient of 9.5 X 10-7 cm2/s. The enzyme was active only at apurinic/apyridiminic sites, regardless of whether they were produced by heating the DNA at acidic pH or by alkylation with the ultimate carcinogen methyl methanesulphonate. Native DNA was not acted upon. U.v.-irradiated DNA and DNA treated with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene were cleaved to an extent related to the extent of apurinic/apyridiminic sites. Enzymic activity was not dependent upon Mg2+, but was stimulated approx. 3-fold by 4mM-Mg2+. The enzyme did not bind to DEAE-cellulose or CM-cellulose at KCl concentrations greater than 160 mM. The endonuclease was obtained free of exonuclease and 3-methyladenine-DNA glycosylase activity in five chromatographic steps.

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Selected References

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

  1. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armel P. R., Wallace S. S. Apurinic endonucleases from Saccharomyces cerevisiae. Nucleic Acids Res. 1978 Sep;5(9):3347–3356. doi: 10.1093/nar/5.9.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bibor V., Verly W. G. Purification and properties of the endonuclease specific for apurinic sites of Bacillus stearothermophilus. J Biol Chem. 1978 Feb 10;253(3):850–855. [PubMed] [Google Scholar]
  4. Braun A., Grossman L. An endonuclease from Escherichia coli that acts preferentially on UV-irradiated DNA and is absent from the uvrA and uvrB mutants. Proc Natl Acad Sci U S A. 1974 May;71(5):1838–1842. doi: 10.1073/pnas.71.5.1838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chlebowicz E., Jachymczyk W. J. Endonuclease for apurinic sites in yeast. Comparison of the enzyme activity in the wild type and in rad mutants of Saccharomyces cerevisiae to MNS. Mol Gen Genet. 1977 Jul 20;154(2):221–223. doi: 10.1007/BF00330841. [DOI] [PubMed] [Google Scholar]
  6. Espejo R. T., Canelo E. S., Sinsheimer R. L. DNA of bacteriophage PM2: a closed circular double-stranded molecule. Proc Natl Acad Sci U S A. 1969 Aug;63(4):1164–1168. doi: 10.1073/pnas.63.4.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fäth W. W., Brendel M. An improved assay of UV-induced thymine-containing dimers in Saccharomyces cerevisiae. Z Naturforsch C. 1975 Nov-Dec;30(6):804–810. doi: 10.1515/znc-1975-11-1220. [DOI] [PubMed] [Google Scholar]
  8. Hecht R., Thielmann H. W. Purification and characterization of an endonuclease from Micrococcus luteus that acts on depurinated and carcinogen-modified DNA. Eur J Biochem. 1978 Sep 1;89(2):607–618. doi: 10.1111/j.1432-1033.1978.tb12565.x. [DOI] [PubMed] [Google Scholar]
  9. Jachymczyk W. J., Chlebowicz E., Swietlinska Z., Zuk J. Alkaline sucrose sedimentation studies of MMS-induced DNA single-strand breakage and rejoining in the wild type and in UV-sensitive mutants of Saccharomyces cerevisiae. Mutat Res. 1977 Apr;43(1):1–10. doi: 10.1016/0027-5107(77)90126-9. [DOI] [PubMed] [Google Scholar]
  10. Kaplan J. C., Kushner S. R., Grossman L. Enzymatic repair of DNA, 1. Purification of two enzymes involved in the excision of thymine dimers from ultraviolet-irradiated DNA. Proc Natl Acad Sci U S A. 1969 May;63(1):144–151. doi: 10.1073/pnas.63.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kirtikar D. M., Goldthwait D. A. The enzymatic release of O6-methylguanine and 3-methyladenine from DNA reacted with the carcinogen N-methyl-N-nitrosourea. Proc Natl Acad Sci U S A. 1974 May;71(5):2022–2026. doi: 10.1073/pnas.71.5.2022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuebler J. P., Goldthwait D. A. An endonuclease from calf liver specific for apurinic sites in DNA. Biochemistry. 1977 Apr 5;16(7):1370–1377. doi: 10.1021/bi00626a021. [DOI] [PubMed] [Google Scholar]
  13. Kuhnlein U., Lee B., Penhoet E. E., Linn S. Xeroderma pigmentosum fibroblasts of the D group lack an apurinic DNA endonuclease species with a low apparent Km. Nucleic Acids Res. 1978 Mar;5(3):951–960. doi: 10.1093/nar/5.3.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LAWLEY P. D., BROOKES P. FURTHER STUDIES ON THE ALKYLATION OF NUCLEIC ACIDS AND THEIR CONSTITUENT NUCLEOTIDES. Biochem J. 1963 Oct;89:127–138. doi: 10.1042/bj0890127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laval J. Two enzymes are required from strand incision in repair of alkylated DNA. Nature. 1977 Oct 27;269(5631):829–832. doi: 10.1038/269829a0. [DOI] [PubMed] [Google Scholar]
  16. Lindahl T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair. Prog Nucleic Acid Res Mol Biol. 1979;22:135–192. doi: 10.1016/s0079-6603(08)60800-4. [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. Linsley W. S., Penhoet E. E., Linn S. Human endonuclease specific for apurinic/apyrimidinic sites in DNA. Partial purification and characterization of multiple forms from placenta. J Biol Chem. 1977 Feb 25;252(4):1235–1242. [PubMed] [Google Scholar]
  19. Ljungquist S., Lindahl T. A mammalian endonuclease specific for apurinic sites in double-stranded deoxyribonucleic acid. I. Purification and general properties. J Biol Chem. 1974 Mar 10;249(5):1530–1535. [PubMed] [Google Scholar]
  20. Ljungquist S., Lindahl T. Relation between Escherichia coli endonucleases specific for apurinic sites in DNA and exonuclease III. Nucleic Acids Res. 1977 Aug;4(8):2871–2879. doi: 10.1093/nar/4.8.2871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ljungquist S., Nyberg B., Lindahl T. Mammalian DNA endonuclease acting at apurinic sites: absence of associated exonuclease activity. FEBS Lett. 1975 Sep 15;57(2):169–171. doi: 10.1016/0014-5793(75)80708-3. [DOI] [PubMed] [Google Scholar]
  22. Lotlikar P. D., Miller E. C., Miller J. A., Margreth A. The enzymatic reduction of the N-hydroxy derivatives of 2-acetylaminofluorene and related carcinogens by tissue preparations. Cancer Res. 1965 Nov;25(10):1743–1752. [PubMed] [Google Scholar]
  23. Ludwig G., Thielmann H. W. Apurinic acid endonuclease activity from mouse epidermal cells. Nucleic Acids Res. 1979 Jun 25;6(8):2901–2917. doi: 10.1093/nar/6.8.2901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  25. Margison G. P., O'Connor P. J. Biological implications of the instability of the N-glycosidic bone of 3-methyldeoxyadenosine in DNA. Biochim Biophys Acta. 1973 Dec 21;331(3):349–356. doi: 10.1016/0005-2787(73)90021-x. [DOI] [PubMed] [Google Scholar]
  26. Nes I. F., Nissen-Meyer J. Endonuclease activities from a permanently established mouse cell line that act upon DNA damaged by ultraviolet light, acid and osmium tetroxide. Biochim Biophys Acta. 1978 Aug 23;520(1):111–121. doi: 10.1016/0005-2787(78)90012-6. [DOI] [PubMed] [Google Scholar]
  27. Seeberg E. Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA+ and uvrB+/uvrC+ gene products. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2569–2573. doi: 10.1073/pnas.75.6.2569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  29. Singer B. The chemical effects of nucleic acid alkylation and their relation to mutagenesis and carcinogenesis. Prog Nucleic Acid Res Mol Biol. 1975;15(0):219–284. [PubMed] [Google Scholar]
  30. Svachulová J., Satava J., Velemínský J. A barley endonuclease specific for apurinic DNA. Isolation and partial characterization. Eur J Biochem. 1978 Jun 15;87(2):215–220. doi: 10.1111/j.1432-1033.1978.tb12368.x. [DOI] [PubMed] [Google Scholar]
  31. Teebor G. W., Duker N. J. Human endonuclease activity for DNA apurinic sites. Nature. 1975 Dec 11;258(5535):544–547. doi: 10.1038/258544a0. [DOI] [PubMed] [Google Scholar]
  32. Thibodeau L., Verly W. G. Purification and properties of a plant endonuclease specific for apurinic sites. J Biol Chem. 1977 May 25;252(10):3304–3309. [PubMed] [Google Scholar]
  33. Thielmann H. W. Detection of strand breaks in phiX 174 RFI and PM2 DNA reacted with ultimate and proximate carcinogens. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 1977 Oct;90(1):37–69. doi: 10.1007/BF00306020. [DOI] [PubMed] [Google Scholar]
  34. Tomura T., Van Lancker J. L. Purification of human and monkey endonucleases acting on ultraviolet-irradiated DNA. Arch Biochem Biophys. 1980 May;201(2):636–639. doi: 10.1016/0003-9861(80)90553-6. [DOI] [PubMed] [Google Scholar]
  35. Verly W. G., Paquette Y. An endonuclease for depurinated DNA in rat liver. Can J Biochem. 1973 Jul;51(7):1003–1009. doi: 10.1139/o73-130. [DOI] [PubMed] [Google Scholar]
  36. Verly W. G., Paquette Y., Thibodeau L. Nuclease for DNA apurinic sites may be involved in the maintenance of DNA in normal cells. Nat New Biol. 1973 Jul 18;244(133):67–69. doi: 10.1038/newbio244067a0. [DOI] [PubMed] [Google Scholar]
  37. Verly W. G., Rassart E. Purification of Escherichia coli endonuclease specific for apurinic sites in DNA. J Biol Chem. 1975 Oct 25;250(20):8214–8219. [PubMed] [Google Scholar]
  38. Vinograd J., Lebowitz J., Radloff R., Watson R., Laipis P. The twisted circular form of polyoma viral DNA. Proc Natl Acad Sci U S A. 1965 May;53(5):1104–1111. doi: 10.1073/pnas.53.5.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Woodworth-Gutai M., Lebowitz J., Kato A. C., Denhardt D. T. Ultraviolet light irradiation of PM2 superhelical DNA. Nucleic Acids Res. 1977;4(5):1243–1256. doi: 10.1093/nar/4.5.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. van Lancker J. L., Tomura T. Purification and some properties of a mammalian repair endonuclease. Biochim Biophys Acta. 1974 Jun 14;353(1):99–114. doi: 10.1016/0005-2787(74)90101-4. [DOI] [PubMed] [Google Scholar]

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