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. 1991 Oct 15;88(20):9127–9131. doi: 10.1073/pnas.88.20.9127

Cloning and characterization of a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to chromosome 16.

L Samson 1, B Derfler 1, M Boosalis 1, K Call 1
PMCID: PMC52665  PMID: 1924375

Abstract

We described previously the isolation of a Saccharomyces cerevisiae 3-methyladenine (3-MeAde) DNA glycosylase repair gene (MAG) by its expression in glycosylase-deficient Escherichia coli alkA tag mutant cells and its ability to rescue these cells from the toxic effects of alkylating agents. Here we extend this cross-species functional complementation approach to the isolation of a full-length human 3-MeAde DNA glycosylase cDNA that rescues alkA tag E. coli from killing by methyl methanesulfonate, and we have mapped the gene to human chromosome 16. The cloned cDNA, expressed from the pBR322 beta-lactamase promoter, contains an 894-base-pair open reading frame encoding a 32,894-Da protein able to release 3-MeAde, but not 7-methylguanine, from alkylated DNA. Surprisingly, the predicted human protein does not share significant amino acid sequence homology with the bacterial AlkA and Tag glycosylases or the yeast MAG glycosylase, but it does share extensive amino acid sequence homology with a rat 3-MeAde DNA glycosylase and significant DNA sequence homology with genes from several mammalian species. The cloning of a human 3-MeAde DNA glycosylase cDNA represents a key step in generating 3-MeAde repair-deficient cells and the determination of the in vivo role of this DNA repair enzyme in protecting against the toxic and carcinogenic effects of alkylating agents.

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Selected References

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

  1. Chen J., Derfler B., Maskati A., Samson L. Cloning a eukaryotic DNA glycosylase repair gene by the suppression of a DNA repair defect in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7961–7965. doi: 10.1073/pnas.86.20.7961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chen J., Derfler B., Samson L. Saccharomyces cerevisiae 3-methyladenine DNA glycosylase has homology to the AlkA glycosylase of E. coli and is induced in response to DNA alkylation damage. EMBO J. 1990 Dec;9(13):4569–4575. doi: 10.1002/j.1460-2075.1990.tb07910.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gallagher P. E., Brent T. P. Partial purification and characterization of 3-methyladenine-DNA glycosylase from human placenta. Biochemistry. 1982 Dec 7;21(25):6404–6409. doi: 10.1021/bi00268a013. [DOI] [PubMed] [Google Scholar]
  4. Gerson S. L., Miller K., Berger N. A. O6 alkylguanine-DNA alkyltransferase activity in human myeloid cells. J Clin Invest. 1985 Dec;76(6):2106–2114. doi: 10.1172/JCI112215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gerson S. L., Trey J. E., Miller K., Benjamin E. Repair of O6-alkylguanine during DNA synthesis in murine bone marrow hematopoietic precursors. Cancer Res. 1987 Jan 1;47(1):89–95. [PubMed] [Google Scholar]
  6. Harper P. S., Frézal J., Ferguson-Smith M. A., Schinzel A. Report of the committee on clinical disorders and chromosomal deletion syndromes. Cytogenet Cell Genet. 1989;51(1-4):563–611. doi: 10.1159/000132808. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. O'Connor T. R., Laval F. Isolation and structure of a cDNA expressing a mammalian 3-methyladenine-DNA glycosylase. EMBO J. 1990 Oct;9(10):3337–3342. doi: 10.1002/j.1460-2075.1990.tb07534.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Prochownik E. V., Markham A. F., Orkin S. H. Isolation of a cDNA clone for human antithrombin III. J Biol Chem. 1983 Jul 10;258(13):8389–8394. [PubMed] [Google Scholar]
  10. Rebeck G. W., Coons S., Carroll P., Samson L. A second DNA methyltransferase repair enzyme in Escherichia coli. Proc Natl Acad Sci U S A. 1988 May;85(9):3039–3043. doi: 10.1073/pnas.85.9.3039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Reeders S. T., Hildebrand C. E. Report of the committee on the genetic constitution of chromosome 16. Cytogenet Cell Genet. 1989;51(1-4):299–318. doi: 10.1159/000132796. [DOI] [PubMed] [Google Scholar]
  12. Samson L., Linn S. DNA alkylation repair and the induction of cell death and sister chromatid exchange in human cells. Carcinogenesis. 1987 Feb;8(2):227–230. doi: 10.1093/carcin/8.2.227. [DOI] [PubMed] [Google Scholar]
  13. Singer B., Brent T. P. Human lymphoblasts contain DNA glycosylase activity excising N-3 and N-7 methyl and ethyl purines but not O6-alkylguanines or 1-alkyladenines. Proc Natl Acad Sci U S A. 1981 Feb;78(2):856–860. doi: 10.1073/pnas.78.2.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Talmadge K., Stahl S., Gilbert W. Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3369–3373. doi: 10.1073/pnas.77.6.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Van Duin M., Hoeijmakers J. H. Cloning of human repair genes by genomic DNA transfection. Ann Ist Super Sanita. 1989;25(1):131–142. [PubMed] [Google Scholar]
  17. Volkert M. R., Nguyen D. C., Beard K. C. Escherichia coli gene induction by alkylation treatment. Genetics. 1986 Jan;112(1):11–26. doi: 10.1093/genetics/112.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Washington W. J., Dunn W. C., Jr, Generoso W. M., Mitra S. Tissue-specific variation in repair activity for 3-methyladenine in DNA in two stocks of mice. Mutat Res. 1988 Mar-Apr;207(3-4):165–169. doi: 10.1016/0165-7992(88)90082-6. [DOI] [PubMed] [Google Scholar]
  20. Winston F., Chumley F., Fink G. R. Eviction and transplacement of mutant genes in yeast. Methods Enzymol. 1983;101:211–228. doi: 10.1016/0076-6879(83)01016-2. [DOI] [PubMed] [Google Scholar]

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