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Study of bacteriophage T4-encoded Dam DNA (adenine-N6)-methyltransferase binding with substrates by rapid laser UV cross-linking.

TitleStudy of bacteriophage T4-encoded Dam DNA (adenine-N6)-methyltransferase binding with substrates by rapid laser UV cross-linking.
Publication TypeJournal Article
Year of Publication2007
AuthorsEvdokimov, AA, Sclavi, B, Zinoviev, VV, Malygin, EG, Hattman, S, Buckle, M
JournalJ Biol Chem
Volume282
Issue36
Pagination26067-76
Date Published2007 Sep 07
ISSN0021-9258
KeywordsDNA, DNA Methylation, Lasers, Models, Molecular, Protein Binding, S-Adenosylhomocysteine, S-Adenosylmethionine, Site-Specific DNA-Methyltransferase (Adenine-Specific), Substrate Specificity, Ultraviolet Rays, Viral Proteins
Abstract

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (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 Nd(3+):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.

DOI10.1074/jbc.M700866200
Alternate JournalJ. Biol. Chem.
PubMed ID17630395
Grant ListTW05755 / TW / FIC NIH HHS / United States