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J. Biol. Chem., Vol. 282, Issue 36, 26067-26076, September 7, 2007

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Study of Bacteriophage T4-encoded Dam DNA (Adenine-N⁶)-methyltransferase Binding with Substrates by Rapid Laser UV Cross-linking^*

Alexey A. Evdokimov¹, Bianca Sclavi, Victor V. Zinoviev, Ernst G. Malygin, Stanley Hattman^¶, and Malcolm Buckle²

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

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N⁶)-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-Lhomocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme·substrate complexes using an Nd³⁺:yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme·substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam·AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.

Received for publication, January 30, 2007 , and in revised form, July 12, 2007.

^* This work was supported in part by United States Public Health Service Grant TW05755 from the Fogarty International Center and Russian Foundation for Basic Research Grant 06-04-49698. 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.

¹ Supported by a short-term fellowship from the Human Frontier Science Program and by Grant MK-1578.2005.4 from the President of Russian Federation for Young Scientists.

² To whom correspondence should be addressed. Tel.: 331-4740-7673; Fax: 331-4740-7671; E-mail: buckle{at}lbpa.ens-cachan.fr.

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