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Nucleic Acids Research, 2000, Vol. 28, No. 21 4207-4211
© 2000 Oxford University Press

Pre-steady state kinetics of bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase: interaction with native (GATC) or modified sites

Ernst G. Malygin, William M. Lindstrom Jr1, Samuel L. Schlagman2, Stanley Hattman2,* and Norbert O. Reich1

Institute of Molecular Biology, State Research Center of Virology and Biotechnology ‘Vector’, Novosibirsk 633159, Russia, 1Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA and 2Department of Biology, University of Rochester, NY 14627-0211, USA

The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine [generating N6-methyladenine and S-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant kmeth for unmethylated and hemimethylated substrates (0.56 and 0.47 s–1, respectively) was at least 20-fold larger than the overall reaction rate constant kcat (0.023 s–1). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit kmeth. Preformed T4 Dam MTase–DNA complexes are less efficient than preformed T4 Dam MTase–AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.

* To whom correspondence should be addressed. Tel: +1 716 274 8046; Fax: +1 716 275 2070; Email: moddna@mail.rochester.edu


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