Abstract
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 (1–4). 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.
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References
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.
Saffhill, R., Margison, G. P., and O’Connor, G. P. (1985) Mechanisms of carcinogenesis induced by alkylating agents. Biochim. Biophys. Acta 823, 111–145.
Yarosh, D. B. (1985) The role of O 6-methylguanine-DNA methyltransferase in cell survival, mutagenesis and carcinogenesis. Mut. Res. 145, 1–16.
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.
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.
Lindahl, T., Demple, B., and Robbins, P. (1982) Suicide inactivation of the E. coli O 6-methylguanine-DNA methyltransferase. Embo J. 1, 1359–1363.
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.
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.
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.
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.
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.
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.).
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.
Instructions—TKO 102 Standards Kit (Hoefer Scientific Instruments, Newcastle-under-Lyme, UK).
Cesarone, C. F., Bolognesi L., and Santi L. (1979) Improved microfluorimetric DNA determination in biological material using 33258 Hoechst. Anal. Biol. 100, 188.
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Watson, A.J., Margison, G.P. (1999). O 6-Alkylguanine-DNA Alkyltransferase Assay. In: Brown, R., Böger-Brown, U. (eds) Cytotoxic Drug Resistance Mechanisms. Methods in Molecular Medicine™, vol 28. Humana Press. https://doi.org/10.1385/1-59259-687-8:167
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DOI: https://doi.org/10.1385/1-59259-687-8:167
Publisher Name: Humana Press
Print ISBN: 978-0-89603-603-1
Online ISBN: 978-1-59259-687-4
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