Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1986 Dec 1;5(12):3195–3200. doi: 10.1002/j.1460-2075.1986.tb04629.x

Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene.

H Kataoka, J Hall, P Karran
PMCID: PMC1167312  PMID: 3545815

Abstract

Dual expression vectors derived from pSV2gpt and encoding all or part of the Escherichia coli ada+ gene have been constructed. Following transformation into an E. coli ada strain or transfection and stable integration into the genome of Chinese hamster ovary (CHO) cells, plasmid vectors containing the whole ada+ gene conferred resistance to both killing and mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thus, the bacterial DNA repair gene was functionally expressed in the mammalian cells. Plasmids containing an N-terminal fragment of the ada+ gene which encoded only one of the two methyltransferase activities of the Ada protein did not significantly protect E. coli or CHO cells against MNNG. These results are consistent with the central role of the intact ada+ gene in controlling the adaptive response to alkylating agents in E. coli. However, the data further suggest that some alkylation lesions in DNA, such as O6-methylguanine, may exert partly different biological effects in E. coli and mammalian cells.

Full text

PDF
3195

Images in this article

3197Fig. 3.
on p.3197

Selected References

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

  1. Baker R. M., Van Voorhis W. C., Spencer L. A. HeLa cell variants that differ in sensitivity to monofunctional alkylating agents, with independence of cytotoxic and mutagenic responses. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5249–5253. doi: 10.1073/pnas.76.10.5249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Bradley W. E., Letovanec D. High-frequency nonrandom mutational event at the adenine phosphoribosyltransferase (aprt) locus of sib-selected CHO variants heterozygous for aprt. Somatic Cell Genet. 1982 Jan;8(1):51–66. doi: 10.1007/BF01538650. [DOI] [PubMed] [Google Scholar]
  4. Chernajovsky Y., Mory Y., Chen L., Marks Z., Novick D., Rubinstein M., Revel M. Efficient constitutive production of human fibroblast interferon by hamster cells transformed with the IFN-beta 1 gene fused to an SV40 early promoter. DNA. 1984 Aug;3(4):297–308. doi: 10.1089/dna.1.1984.3.297. [DOI] [PubMed] [Google Scholar]
  5. Day R. S., 3rd, Ziolkowski C. H., Scudiero D. A., Meyer S. A., Lubiniecki A. S., Girardi A. J., Galloway S. M., Bynum G. D. Defective repair of alkylated DNA by human tumour and SV40-transformed human cell strains. Nature. 1980 Dec 25;288(5792):724–727. doi: 10.1038/288724a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Domoradzki J., Pegg A. E., Dolan M. E., Maher V. M., McCormick J. J. Depletion of O6-methylguanine-DNA-methyltransferase in human fibroblasts increases the mutagenic response to N-methyl-N'-nitro-N-nitrosoguanidine. Carcinogenesis. 1985 Dec;6(12):1823–1826. doi: 10.1093/carcin/6.12.1823. [DOI] [PubMed] [Google Scholar]
  8. Evensen G., Seeberg E. Adaptation to alkylation resistance involves the induction of a DNA glycosylase. Nature. 1982 Apr 22;296(5859):773–775. doi: 10.1038/296773a0. [DOI] [PubMed] [Google Scholar]
  9. Friedman J., Huberman E. Postreplication repair and the susceptibility of Chinese hamster cells to cytotoxic and mutagenic effects of alkylating agents. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6072–6076. doi: 10.1073/pnas.77.10.6072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harris A. L., Karran P., Lindahl T. O6-Methylguanine-DNA methyltransferase of human lymphoid cells: structural and kinetic properties and absence in repair-deficient cells. Cancer Res. 1983 Jul;43(7):3247–3252. [PubMed] [Google Scholar]
  11. Hill-Perkins M., Jones M. D., Karran P. Site-specific mutagenesis in vivo by single methylated or deaminated purine bases. Mutat Res. 1986 Sep;162(2):153–163. doi: 10.1016/0027-5107(86)90081-3. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Jeggo P. Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents. J Bacteriol. 1979 Sep;139(3):783–791. doi: 10.1128/jb.139.3.783-791.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karran P., Hjelmgren T., Lindahl T. Induction of a DNA glycosylase for N-methylated purines is part of the adaptive response to alkylating agents. Nature. 1982 Apr 22;296(5859):770–773. doi: 10.1038/296770a0. [DOI] [PubMed] [Google Scholar]
  15. Karran P., Lindahl T., Griffin B. Adaptive response to alkylating agents involves alteration in situ of O6-methylguanine residues in DNA. Nature. 1979 Jul 5;280(5717):76–77. doi: 10.1038/280076a0. [DOI] [PubMed] [Google Scholar]
  16. Karran P. Possible depletion of a DNA repair enzyme in human lymphoma cells by subversive repair. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5285–5289. doi: 10.1073/pnas.82.16.5285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karran P., Williams S. A. The cytotoxic and mutagenic effects of alkylating agents on human lymphoid cells are caused by different DNA lesions. Carcinogenesis. 1985 May;6(5):789–792. doi: 10.1093/carcin/6.5.789. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kuzmich S., Marky L. A., Jones R. A. Specifically alkylated DNA fragments. Synthesis and physical characterization of d[CGC(O6Me)GCG] and d[CGT(O6Me)GCG]. Nucleic Acids Res. 1983 May 25;11(10):3393–3403. doi: 10.1093/nar/11.10.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lawley P. D. Some chemical aspects of dose-response relationships in alkylation mutagenesis. Mutat Res. 1974 Jun;23(3):283–295. doi: 10.1016/0027-5107(74)90102-x. [DOI] [PubMed] [Google Scholar]
  22. Loechler E. L., Green C. L., Essigmann J. M. In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6271–6275. doi: 10.1073/pnas.81.20.6271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McCarthy T. V., Karran P., Lindahl T. Inducible repair of O-alkylated DNA pyrimidines in Escherichia coli. EMBO J. 1984 Mar;3(3):545–550. doi: 10.1002/j.1460-2075.1984.tb01844.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCarthy T. V., Lindahl T. Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E. coli. Nucleic Acids Res. 1985 Apr 25;13(8):2683–2698. doi: 10.1093/nar/13.8.2683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Meuth M. Role of deoxynucleoside triphosphate pools in the cytotoxic and mutagenic effects of DNA alkylating agents. Somatic Cell Genet. 1981 Jan;7(1):89–102. doi: 10.1007/BF01544750. [DOI] [PubMed] [Google Scholar]
  26. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  27. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nalbantoglu J., Goncalves O., Meuth M. Structure of mutant alleles at the aprt locus of Chinese hamster ovary cells. J Mol Biol. 1983 Jul 5;167(3):575–594. doi: 10.1016/s0022-2836(83)80099-0. [DOI] [PubMed] [Google Scholar]
  29. Newbold R. F., Warren W., Medcalf A. S., Amos J. Mutagenicity of carcinogenic methylating agents is associated with a specific DNA modification. Nature. 1980 Feb 7;283(5747):596–599. doi: 10.1038/283596a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Sedgwick B. Molecular cloning of a gene which regulates the adaptive response to alkylating agents in Escherichia coli. Mol Gen Genet. 1983;191(3):466–472. doi: 10.1007/BF00425764. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Shooter K. V. DNA phosphotriesters as indicators of cumulative carcinogen-induced damage. Nature. 1978 Aug 10;274(5671):612–614. doi: 10.1038/274612a0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Snow E. T., Foote R. S., Mitra S. Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication. J Biol Chem. 1984 Jul 10;259(13):8095–8100. [PubMed] [Google Scholar]
  36. Teo I., Sedgwick B., Demple B., Li B., Lindahl T. Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage. EMBO J. 1984 Sep;3(9):2151–2157. doi: 10.1002/j.1460-2075.1984.tb02105.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]
  40. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES