O6-Alkylguanine-DNA Alkyltransferase Assay

  • Amanda J. Watson
  • Geoffrey P. Margison
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 28)


Alkylating agents exert a wide range of biological effects in both pro- and eukaryotes and there is ever increasing evidence that these effects are mediated via alkylation at the O 6position of guanine in DNA (1, 2, 3, 4). Repair of such adducts can be mediated by O 6-alkylguanine-DNA alkyltransferase (ATase) (3,4). Both pro- and eukaryote ATases transfer alkyl groups from the O 6-position of guanine in alkylated DNA (or from other low molecular weight substrates); (5) to a cysteine residue located at the active site of the protein: the reaction is stoichiometric and the protein is autoinactivated (6). This mechanism has been exploited in the design of several different radioactivity-based assays for the enzyme. These involve either measurement of methyl group transfer to protein or the analysis (e.g., by HPLC) of methylated substrate DNA before and after exposure to cell or tissue extracts or restriction endonuclease (RE) site deprotection of synthetic oligonucleotide substrates containing O 6-methylguanine.


Plateau Level Fume Cupboard Clean Glass Vial Water Vacuum Substrate Specific Activity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Singer, B. (1979) N-nitroso alkylating agents: formation and persistence of alkyl derivatives in mammalian nucleic acids as contributing factors in carcinogenesis. J. Natl. Cancer Inst. 62, 1329–1339.PubMedGoogle Scholar
  2. 2.
    Saffhill, R., Margison, G. P., and O’Connor, G. P. (1985) Mechanisms of carcinogenesis induced by alkylating agents. Biochim. Biophys. Acta 823, 111–145.PubMedGoogle Scholar
  3. 3.
    Yarosh, D. B. (1985) The role of O 6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. Mut. Res. 145, 1–16.Google Scholar
  4. 4.
    Margison, G. P. and O’Connor, P. J. (1990) Biological consequences of reactions with DNA: role of specific lesions, in Handbook of Experimental Pharmacology (Grover, C. S. and Grover, P. L., eds.), pp. 547–571.Google Scholar
  5. 5.
    Dolan, M. E., Moschel, R. C., and Pegg, A. E. (1990) Depletion of mammalian O 6-alkylguanine-DNA alkyltransferase activity by O 6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents. Proc. Natl. Acad. Sci. USA 87, 5368–5372.PubMedCrossRefGoogle Scholar
  6. 6.
    Lindahl, T., Demple, B., and Robbins, P. (1982) Suicide inactivation of the E. coli O 6-methylguanine-DNA methyltransferase. Embo J. 1, 1359–1363.PubMedGoogle Scholar
  7. 7.
    Fan, C. Y., Potter, P. M., Rafferty, J., Watson, A. J., Cawkwell, L., Searle, P. F., O’Connor, P. J., and Margison G. P. (1990) Expression of a human O 6-alkylguanine-DNA-alkyltransferase cDNA in human cells and transgenic mice. Nucleic Acids Res. 18, 5723–5727.PubMedCrossRefGoogle Scholar
  8. 8.
    Margison, G. P., Cooper, D. P., and Brennand, J. (1985) Cloning of the E. coli O 6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay. Nucleic Acids Res. 13, 1939–1952.PubMedCrossRefGoogle Scholar
  9. 9.
    Wilkinson, M. C., Potter, P. M., Cawkwell, L. C., Georgiadis, P., Patel, D., Swann, P. F., and Margison, G. P. (1989) Purification of the E.coli ogt gene product to homogeneity and its rate of action on O 6-methylguanine, O 6-ethylguanine and O 4-methylguanine in dodecadeoxyribonucleotides. Nucleic Acids Res. 17, 8475–8484.PubMedCrossRefGoogle Scholar
  10. 10.
    Wu, R. S., Hurst-Calderone, S., and Kohn, K. W. (1987) Measurement of O 6-alkylguanine-DNA-alkyltransferase activity in human cells and tumour tissues by restriction endonuclease inhibition. Cancer Res. 47, 6229–6235.PubMedGoogle Scholar
  11. 11.
    Brent, T. P. and Remack, J. S. (1988) Formation of covalent complexes between human O 6-alkylguanine-DNA alkyltransferase and BCNU-treated defined length synthetic oligonucleotides. Nucleic Acids Res. 16, 6779–6788.PubMedCrossRefGoogle Scholar
  12. 12.
    Coutinho, L. H., Gilleece, M. H., Dewynter, E. A., Will, A., and Testa, N. G. (1993) Clonal and long-term culture using human bone-marrow, in Haemopoesis: A Practical Approach, vol. 288. (Testa, N. G. and Molyneux, G. M., eds.).Google Scholar
  13. 13.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248–272.PubMedCrossRefGoogle Scholar
  14. 13.
    Instructions—TKO 102 Standards Kit (Hoefer Scientific Instruments, Newcastle-under-Lyme, UK).Google Scholar
  15. 14.
    Cesarone, C. F., Bolognesi L., and Santi L. (1979) Improved microfluorimetric DNA determination in biological material using 33258 Hoechst. Anal. Biol. 100, 188.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Amanda J. Watson
    • 1
  • Geoffrey P. Margison
    • 1
  1. 1.Department of Carcinogenesis, Paterson Institute for Cancer ResearchChristie Hospital NHS TrustManchesterUK

Personalised recommendations