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J. Biol. Chem., Vol. 279, Issue 48, 50012-50018, November 26, 2004

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Bacteriophage T4Dam DNA-(Adenine-N⁶)-methyltransferase

COMPARISON OF PRE-STEADY STATE AND SINGLE TURNOVER METHYLATION OF 40-MER DUPLEXES CONTAINING TWO (UN)MODIFIED TARGET SITES^*

Ernst G. Malygin, Bianca Sclavi¶, Victor V. Zinoviev, Alexey A. Evdokimov||, Stanley Hattman**, and Malcolm Buckle¶

From the Institute of Molecular Biology, State Research Center of Virology and Biotechnology "Vector," Novosibirsk 630559, Russia, the ^**Department of Biology, University of Rochester, Rochester, New York 14627-0211, and the ^¶Enzymologie et Cinetique Structurale, Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquées (UMR 8113 du CNRS) Ecole Normale Superieur de Cachan, 94235 CACHAN, France

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N⁶)-methyltransferase-mediated methyl group transfer from S-adenosyl-L-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-L-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex. Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps. With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase. Single turnover methylation of 40-mer duplexes also proceeded in two stages. First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a two-step methylation. Instead of processive movement of T4Dam, a conformational adaptation occurred. We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand. This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. Under single turnover conditions, there was complete methylation of all target adenine residues with each of the two-site 40-mer duplexes.

Received for publication, August 25, 2004 , and in revised form, September 14, 2004.

^* This work was partly supported by a U. S. Public Health Service grant from the Fogarty International Center (Grant R03 TW05755) and by a U. S. Public Health Service (Grant GM29227) (to S. H.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the French embassy in Russia A. A. within the framework of assistance to Franco-Russian co-operation in 2003.

^|| Supported by a fellowship from the Human Frontier Science Program in 2004.

To whom correspondence should be addressed. Tel.: 585-275-8046; Fax: 585-275-2070; E-mail: modDNA{at}mail.rochester.edu.

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