Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Oct 25;19(20):5597–5601. doi: 10.1093/nar/19.20.5597

Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase.

K Sakumi 1, A Shiraishi 1, H Hayakawa 1, M Sekiguchi 1
PMCID: PMC328962  PMID: 1945835

Abstract

cDNA for O6-methylguanine-DNA methyltransferase was isolated by screening rat liver cDNA libraries, using as a probe the human cDNA sequence for methyltransferase. The rat cDNA encodes a protein with 209 amino acid residues. The predicted amino acid sequence of the rat methyltransferase exhibits considerable homology with those of the human, yeast and bacterial enzymes, especially around putative methyl acceptor sites. When the cDNA was placed under control of the lac promoter and expressed in methyltransferase-deficient Escherichia coli (ada-, ogt-) cells, a characteristic methyltransferase protein was produced. The rat DNA methyltransferase thus expressed could complement the biological defects of the E. coli cell caused by lack of its own DNA methyltransferases; e.g. increased sensitivity to alkylating agents in terms of both cell death and mutation induction.

Full text

PDF
5597

Images in this article

5599Image
on p.5599

Selected References

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

  1. Coulondre C., Miller J. H. Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli. J Mol Biol. 1977 Dec 15;117(3):577–606. doi: 10.1016/0022-2836(77)90059-6. [DOI] [PubMed] [Google Scholar]
  2. Demple B., Jacobsson A., Olsson M., Robins P., Lindahl T. Repair of alkylated DNA in Escherichia coli. Physical properties of O6-methylguanine-DNA methyltransferase. J Biol Chem. 1982 Nov 25;257(22):13776–13780. [PubMed] [Google Scholar]
  3. Demple B., Sedgwick B., Robins P., Totty N., Waterfield M. D., Lindahl T. Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis. Proc Natl Acad Sci U S A. 1985 May;82(9):2688–2692. doi: 10.1073/pnas.82.9.2688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eadie J. S., Conrad M., Toorchen D., Topal M. D. Mechanism of mutagenesis by O6-methylguanine. Nature. 1984 Mar 8;308(5955):201–203. doi: 10.1038/308201a0. [DOI] [PubMed] [Google Scholar]
  5. Gordon A. J., Burns P. A., Glickman B. W. N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli. Mutat Res. 1988 Sep;201(1):219–228. doi: 10.1016/0027-5107(88)90129-7. [DOI] [PubMed] [Google Scholar]
  6. Goth R., Rajewsky M. F. Persistence of O6-ethylguanine in rat-brain DNA: correlation with nervous system-specific carcinogenesis by ethylnitrosourea. Proc Natl Acad Sci U S A. 1974 Mar;71(3):639–643. doi: 10.1073/pnas.71.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hayakawa H., Koike G., Sekiguchi M. Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA. J Mol Biol. 1990 Jun 20;213(4):739–747. doi: 10.1016/S0022-2836(05)80260-8. [DOI] [PubMed] [Google Scholar]
  8. Kleihues P., Margison G. P. Carcinogenicity of N-methyl-N-nitrosourea: possible role of excision repair of O6-methylguanine from DNA. J Natl Cancer Inst. 1974 Dec;53(6):1839–1841. [PubMed] [Google Scholar]
  9. Kodama K. I., Nakabeppu Y., Sekiguchi M. Cloning and expression of the Bacillus subtilis methyltransferase gene in Escherichia coli ada- cells. Mutat Res. 1989 Sep;218(2):153–163. doi: 10.1016/0921-8777(89)90022-0. [DOI] [PubMed] [Google Scholar]
  10. Koike G., Maki H., Takeya H., Hayakawa H., Sekiguchi M. Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase. J Biol Chem. 1990 Sep 5;265(25):14754–14762. [PubMed] [Google Scholar]
  11. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laval F., Laval J. Adaptive response in mammalian cells: crossreactivity of different pretreatments on cytotoxicity as contrasted to mutagenicity. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1062–1066. doi: 10.1073/pnas.81.4.1062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Loveless A. Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature. 1969 Jul 12;223(5202):206–207. doi: 10.1038/223206a0. [DOI] [PubMed] [Google Scholar]
  15. Major G. N., Gardner E. J., Carne A. F., Lawley P. D. Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine. Nucleic Acids Res. 1990 Mar 25;18(6):1351–1359. doi: 10.1093/nar/18.6.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Margison G. P., Butler J., Hoey B. O6-Methylguanine methyltransferase activity is increased in rat tissues by ionising radiation. Carcinogenesis. 1985 Dec;6(12):1699–1702. doi: 10.1093/carcin/6.12.1699. [DOI] [PubMed] [Google Scholar]
  17. Morohoshi F., Hayashi K., Munakata N. Bacillus subtilis ada operon encodes two DNA alkyltransferases. Nucleic Acids Res. 1990 Sep 25;18(18):5473–5480. doi: 10.1093/nar/18.18.5473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nakabeppu Y., Kondo H., Kawabata S., Iwanaga S., Sekiguchi M. Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase. J Biol Chem. 1985 Jun 25;260(12):7281–7288. [PubMed] [Google Scholar]
  19. Nakabeppu Y., Mine Y., Sekiguchi M. Regulation of expression of the cloned ada gene in Escherichia coli. Mutat Res. 1985 Sep;146(2):155–167. doi: 10.1016/0167-8817(85)90006-9. [DOI] [PubMed] [Google Scholar]
  20. Olsson M., Lindahl T. Repair of alkylated DNA in Escherichia coli. Methyl group transfer from O6-methylguanine to a protein cysteine residue. J Biol Chem. 1980 Nov 25;255(22):10569–10571. [PubMed] [Google Scholar]
  21. Pegg A. E., Wiest L., Foote R. S., Mitra S., Perry W. Purification and properties of O6-methylguanine-DNA transmethylase from rat liver. J Biol Chem. 1983 Feb 25;258(4):2327–2333. [PubMed] [Google Scholar]
  22. Potter P. M., Rafferty J. A., Cawkwell L., Wilkinson M. C., Cooper D. P., O'Connor P. J., Margison G. P. Isolation and cDNA cloning of a rat O6-alkylguanine-DNA-alkyltransferase gene, molecular analysis of expression in rat liver. Carcinogenesis. 1991 Apr;12(4):727–733. doi: 10.1093/carcin/12.4.727. [DOI] [PubMed] [Google Scholar]
  23. Potter P. M., Wilkinson M. C., Fitton J., Carr F. J., Brennand J., Cooper D. P., Margison G. P. Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli. Nucleic Acids Res. 1987 Nov 25;15(22):9177–9193. doi: 10.1093/nar/15.22.9177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rahden-Staron I., Laval F. cDNA cloning of the rat O6-methylguanine-DNA-methyltransferase. Biochem Biophys Res Commun. 1991 Jun 14;177(2):597–602. doi: 10.1016/0006-291x(91)91830-6. [DOI] [PubMed] [Google Scholar]
  25. Rydberg B., Spurr N., Karran P. cDNA cloning and chromosomal assignment of the human O6-methylguanine-DNA methyltransferase. cDNA expression in Escherichia coli and gene expression in human cells. J Biol Chem. 1990 Jun 5;265(16):9563–9569. [PubMed] [Google Scholar]
  26. Sassanfar M., Samson L. Identification and preliminary characterization of an O6-methylguanine DNA repair methyltransferase in the yeast Saccharomyces cerevisiae. J Biol Chem. 1990 Jan 5;265(1):20–25. [PubMed] [Google Scholar]
  27. Sklar R., Strauss B. Removal of O6-methylguanine from DNA of normal and xeroderma pigmentosum-derived lymphoblastoid lines. Nature. 1981 Jan 29;289(5796):417–420. doi: 10.1038/289417a0. [DOI] [PubMed] [Google Scholar]
  28. Sukumar S., Notario V., Martin-Zanca D., Barbacid M. Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature. 1983 Dec 15;306(5944):658–661. doi: 10.1038/306658a0. [DOI] [PubMed] [Google Scholar]
  29. Takano K., Nakabeppu Y., Sekiguchi M. Functional sites of the Ada regulatory protein of Escherichia coli. Analysis by amino acid substitutions. J Mol Biol. 1988 May 20;201(2):261–271. doi: 10.1016/0022-2836(88)90137-4. [DOI] [PubMed] [Google Scholar]
  30. Takano K., Nakamura T., Sekiguchi M. Roles of two types of O6-methylguanine-DNA methyltransferases in DNA repair. Mutat Res. 1991 Jan;254(1):37–44. doi: 10.1016/0921-8777(91)90038-q. [DOI] [PubMed] [Google Scholar]
  31. Tano K., Shiota S., Collier J., Foote R. S., Mitra S. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. Proc Natl Acad Sci U S A. 1990 Jan;87(2):686–690. doi: 10.1073/pnas.87.2.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Xiao W., Derfler B., Chen J., Samson L. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene. EMBO J. 1991 Aug;10(8):2179–2186. doi: 10.1002/j.1460-2075.1991.tb07753.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yarosh D. B., Foote R. S., Mitra S., Day R. S., 3rd Repair of O6-methylguanine in DNA by demethylation is lacking in Mer- human tumor cell strains. Carcinogenesis. 1983;4(2):199–205. doi: 10.1093/carcin/4.2.199. [DOI] [PubMed] [Google Scholar]
  34. von Wronski M. A., Shiota S., Tano K., Mitra S., Bigner D. D., Brent T. P. Structural and immunological comparison of indigenous human O6-methylguanine-DNA methyltransferase with that encoded by a cloned cDNA. J Biol Chem. 1991 Jan 15;266(2):1064–1070. [PubMed] [Google Scholar]

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

RESOURCES