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Nucleic Acids Research, 2001, Vol. 29, No. 11 2361-2369
© 2001 Oxford University Press

A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase

Ernst G. Malygin, Alexey A. Evdokimov, Victor V. Zinoviev, Lidiya G. Ovechkina, William M. Lindstrom1, Norbert O. Reich1, Samuel L. Schlagman2 and Stanley Hattman2,*

Institute of Molecular Biology, State Research Center of Virology and Biotechnology ‘Vector’, Novosibirsk 633159, Russia, 1Department of Chemistry, University of California, Santa Barbara, CA 93103, USA and 2Department of Biology, University of Rochester, New York, NY 14627-0211, USA

The fluorescence of 2-aminopurine (2A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target (2A/A duplex) or its methylated derivative (2A/mA duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with 2A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an 2A/2A double-substituted duplex. Since the 2A/mA duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric 2A/A and 2A/mA duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.

* To whom correspondence should be addressed. Tel: +1 716 275 8046; Fax: +1 716 275 2070; Email: moddna{at}mail.rochester.edu


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