Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Oct;176(20):6255–6261. doi: 10.1128/jb.176.20.6255-6261.1994

The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity.

B J Chen 1, P Carroll 1, L Samson 1
PMCID: PMC196966  PMID: 7928996

Abstract

Escherichia coli can ameliorate the toxic effects of alkylating agents either by preventing DNA alkylation or by repairing DNA alkylation damage. The alkylation-sensitive phenotype of E. coli alkB mutants marks the alkB pathway as an extremely effective defense mechanism against the cytotoxic effects of the SN2, but not the SN1, alkylating agents. Although it is clear that AlkB helps cells to better handle alkylated DNA, no DNA alkylation repair function could be assigned to the purified AlkB protein, suggesting that AlkB either acts as part of a complex or acts to regulate the expression of other genes whose products are directly responsible for alkylation resistance. However, here we present evidence that the provision of alkylation resistance is an intrinsic function of the AlkB protein per se. We expressed the E. coli AlkB protein in two human cell lines and found that it confers the same characteristic alkylation-resistant phenotype in this foreign environment as it does in E. coli. AlkB expression rendered human cells extremely resistant to cell killing by the SN2 but not the SN1 alkylating agents but did not affect the ability of dimethyl sulfate (an SN2 agent) to alkylate the genome. We infer that SN2 agents produce a class of DNA damage that is not efficiently produced by SN1 agents and that AlkB somehow prevents this damage from killing the cell.

Full text

PDF
6255

Images in this article

6258Image
on p.6258

Selected References

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

  1. Antonucci T. K., Wen P., Rutter W. J. Eukaryotic promoters drive gene expression in Escherichia coli. J Biol Chem. 1989 Oct 25;264(30):17656–17659. [PubMed] [Google Scholar]
  2. Banga S. S., Boyd J. B., Valerie K., Harris P. V., Kurz E. M., de Riel J. K. denV gene of bacteriophage T4 restores DNA excision repair to mei-9 and mus201 mutants of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1989 May;86(9):3227–3231. doi: 10.1073/pnas.86.9.3227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrows L. R., Borchers A. H., Paxton M. B. Transfectant CHO cells expressing O6-alkylguanine-DNA-alkyltransferase display increased resistance to DNA damage other than O6-guanine alkylation. Carcinogenesis. 1987 Dec;8(12):1853–1859. doi: 10.1093/carcin/8.12.1853. [DOI] [PubMed] [Google Scholar]
  4. Blochlinger K., Diggelmann H. Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells. Mol Cell Biol. 1984 Dec;4(12):2929–2931. doi: 10.1128/mcb.4.12.2929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brennand J., Margison G. P. Reduction of the toxicity and mutagenicity of alkylating agents in mammalian cells harboring the Escherichia coli alkyltransferase gene. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6292–6296. doi: 10.1073/pnas.83.17.6292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Day R. S., 3rd, Babich M. A., Yarosh D. B., Scudiero D. A. The role of O6-methylguanine in human cell killing, sister chromatid exchange induction and mutagenesis: a review. J Cell Sci Suppl. 1987;6:333–353. doi: 10.1242/jcs.1984.supplement_6.22. [DOI] [PubMed] [Google Scholar]
  8. Harris L. C., Margison G. P. Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas. Br J Cancer. 1993 Jun;67(6):1196–1202. doi: 10.1038/bjc.1993.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Ishizaki K., Tsujimura T., Yawata H., Fujio C., Nakabeppu Y., Sekiguchi M., Ikenaga M. Transfer of the E. coli O6-methylguanine methyltransferase gene into repair-deficient human cells and restoration of cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine. Mutat Res. 1986 Sep;166(2):135–141. doi: 10.1016/0167-8817(86)90011-8. [DOI] [PubMed] [Google Scholar]
  11. Kaina B., Fritz G., Mitra S., Coquerelle T. Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents. Carcinogenesis. 1991 Oct;12(10):1857–1867. doi: 10.1093/carcin/12.10.1857. [DOI] [PubMed] [Google Scholar]
  12. Kaina B., Lohrer H., Karin M., Herrlich P. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2710–2714. doi: 10.1073/pnas.87.7.2710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kataoka H., Hall J., Karran P. Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene. EMBO J. 1986 Dec 1;5(12):3195–3200. doi: 10.1002/j.1460-2075.1986.tb04629.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kataoka H., Sekiguchi M. Molecular cloning and characterization of the alkB gene of Escherichia coli. Mol Gen Genet. 1985;198(2):263–269. doi: 10.1007/BF00383004. [DOI] [PubMed] [Google Scholar]
  15. Kataoka H., Yamamoto Y., Sekiguchi M. A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate. J Bacteriol. 1983 Mar;153(3):1301–1307. doi: 10.1128/jb.153.3.1301-1307.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kondo H., Nakabeppu Y., Kataoka H., Kuhara S., Kawabata S., Sekiguchi M. Structure and expression of the alkB gene of Escherichia coli related to the repair of alkylated DNA. J Biol Chem. 1986 Nov 25;261(33):15772–15777. [PubMed] [Google Scholar]
  17. Lawley P. D., Thatcher C. J. Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N'-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation. Biochem J. 1970 Feb;116(4):693–707. doi: 10.1042/bj1160693. [DOI] [PMC free article] [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. Pegg A. E. Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res. 1990 Oct 1;50(19):6119–6129. [PubMed] [Google Scholar]
  20. 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]
  21. Rebeck G. W., Samson L. Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase. J Bacteriol. 1991 Mar;173(6):2068–2076. doi: 10.1128/jb.173.6.2068-2076.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Samson L. D. The repair of DNA alkylation damage by methyltransferases and glycosylases. Essays Biochem. 1992;27:69–78. [PubMed] [Google Scholar]
  23. Samson L., Cairns J. A new pathway for DNA repair in Escherichia coli. Nature. 1977 May 19;267(5608):281–283. doi: 10.1038/267281a0. [DOI] [PubMed] [Google Scholar]
  24. Samson L., Derfler B., Boosalis M., Call K. Cloning and characterization of a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to chromosome 16. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9127–9131. doi: 10.1073/pnas.88.20.9127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Samson L., Derfler B., Waldstein E. A. Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5607–5610. doi: 10.1073/pnas.83.15.5607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Samson L. The suicidal DNA repair methyltransferases of microbes. Mol Microbiol. 1992 Apr;6(7):825–831. doi: 10.1111/j.1365-2958.1992.tb01533.x. [DOI] [PubMed] [Google Scholar]
  28. Sedgwick B., Robins P. Isolation of mutants of Escherichia coli with increased resistance to alkylating agents: mutants deficient in thiols and mutants constitutive for the adaptive response. Mol Gen Genet. 1980;180(1):85–90. doi: 10.1007/BF00267355. [DOI] [PubMed] [Google Scholar]
  29. Shevell D. E., Friedman B. M., Walker G. C. Resistance to alkylation damage in Escherichia coli: role of the Ada protein in induction of the adaptive response. Mutat Res. 1990 Nov-Dec;233(1-2):53–72. doi: 10.1016/0027-5107(90)90151-s. [DOI] [PubMed] [Google Scholar]
  30. Teo I., Sedgwick B., Kilpatrick M. W., McCarthy T. V., Lindahl T. The intracellular signal for induction of resistance to alkylating agents in E. coli. Cell. 1986 Apr 25;45(2):315–324. doi: 10.1016/0092-8674(86)90396-x. [DOI] [PubMed] [Google Scholar]
  31. Valerie K., Green A. P., de Riel J. K., Henderson E. E. Transient and stable complementation of ultraviolet repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4. Cancer Res. 1987 Jun 1;47(11):2967–2971. [PubMed] [Google Scholar]
  32. Valerie K., de Riel J. K., Henderson E. E. Genetic complementation of UV-induced DNA repair in Chinese hamster ovary cells by the denV gene of phage T4. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7656–7660. doi: 10.1073/pnas.82.22.7656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Volkert M. R., Hajec L. I. Molecular analysis of the aidD6::Mu d1 (bla lac) fusion mutation of Escherichia coli K12. Mol Gen Genet. 1991 Oct;229(2):319–323. doi: 10.1007/BF00272173. [DOI] [PubMed] [Google Scholar]
  34. Volkert M. R., Nguyen D. C. Induction of specific Escherichia coli genes by sublethal treatments with alkylating agents. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4110–4114. doi: 10.1073/pnas.81.13.4110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Xiao W., Samson L. The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions. Nucleic Acids Res. 1992 Jul 25;20(14):3599–3606. doi: 10.1093/nar/20.14.3599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yates J., Warren N., Reisman D., Sugden B. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3806–3810. doi: 10.1073/pnas.81.12.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. von Hofe E., Fairbairn L., Margison G. P. Relationship between O6-alkylguanine-DNA alkyltransferase activity and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation, transformation, and cytotoxicity in C3H/10T1/2 cells expressing exogenous alkyltransferase genes. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11199–11203. doi: 10.1073/pnas.89.23.11199. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES