title: Differential methylation kinetics of individual target site strands by T4Dam DNA methyltransferase.
abstract: Prokaryote DNA methyltransferases (MTases) of the Dam family (including those of bacteriophages T2 and T4) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. Dam DNA MTases, as all site-specific enzymes interacting with polymeric DNA, require a mechanism of action that ensures a rapid search for specific targets for catalytic action, during both the initial and subsequent rounds of methylation. The results of pre-steady-state (reaction burst) and steady-state methylation analyses of individual targets permitted us to monitor the action of T4Dam, which has three degrees of freedom: sliding, reorientation and adaptation to the canonical GATC sequence. The salient results are as follows: (i) 40mer substrate duplexes containing two canonical GATC sites showed differential methylation of the potential targets, i.e., T4Dam exhibited a preference for one site/target, which may present the better 'kinetic trap' for the enzyme. (ii) Prior hemimethylation of the two sites made both targets equally capable of being methylated during the pre-steady-state reaction. (iii) Although capable of moving in either direction along double-stranded DNA, there are some restrictions on T4Dam reorientation/adaptation on 40mer duplexes.
authors.affiliation: State Research Center of Virology and Biotechnology Vector, Novosibirsk, Russia.
authors.forename: Victor V
authors.initials: VV
authors.lastname: Zinoviev
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Evdokimov
authors.forename: Ernst G
authors.initials: EG
authors.lastname: Malygin
authors.forename: Bianca
authors.initials: B
authors.lastname: Sclavi
authors.forename: Malcolm
authors.initials: M
authors.lastname: Buckle
authors.forename: Stanley
authors.initials: S
authors.lastname: Hattman
mesh_terms: D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D007700:Kinetics; D013379:Substrate Specificity
chemical_list: D015254:DNA Modification Methylases