Alexey A. Evdokimov

Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase: Processivity and orientation to the methylation target

We carried out steady state and pre-steady state (burst) kinetic analyses of the bacteriophage T4 Dam DNA-(N 6-adenine)-methyltransferase (MTase)-mediated methyl group transfer fromS-adenosyl-l-methionine (AdoMet) to Ade in oligonucleotide duplexes containing one or two specific GATC sites with different combinations of methylated and unmodified targets. We compared the results for ligated 40-mer duplexes with those of the mixtures of the two unligated duplexes used to generate the 40-mers. The salient results are as follows: (i) T4 Dam MTase modifies 40-mer duplexes in a processive fashion. (ii) During processive movement, T4 Dam rapidly exchanges productS-adenosyl-l-homocysteine (AdoHcy) for substrate AdoMet without dissociating from the DNA duplex. (iii) T4 Dam processivity is consistent with an ordered bi-bi mechanism AdoMet↓DNA↓DNAMe↑AdoHcy↑. However, in contrast to the steady state, here DNAMe↑ signifies departure from a methylated site GMTC↑ without physically dissociating from the DNA. (iv) Following methyl transfer at one site and linear diffusion to a hemimethylated site, a reconstituted T4 Dam-AdoMet complex rapidly reorients itself to the (productive) unmethylated strand. T4 Dam-AdoHcy cannot reorient at an enzymatically created GMTC site. (v) The inhibition potential of fully methylated sites 5′-GMTC/5′-GMTC is much lower for a long DNA molecule compared with short single-site duplexes.

Dimeric/oligomeric DNA methyltransferases: an unfinished story.

Abstract DNA methyltransferases (MTases) are enzymes that carry out post-replicative sequence-specific modifications. The initial experimental data on the structure and kinetic characteristics of the EcoRI MTase led to the paradigm that type II systems comprise dimeric endonucleases and monomeric MTases. In retrospect, this was logical because, while the biological substrate of the restriction endonuclease is two-fold symmetrical, the in vivo substrate for the MTase is generally hemi-methylated and, hence, inherently asymmetric. Thus, the paradigm was extended to include all DNA MTases except the more complex bifunctional type I and type III enzymes. Nevertheless, a gradual enlightenment grew over the last decade that has changed the accepted view on the structure of DNA MTases. These results necessitate a more complex view of the structure and function of these important enzymes.

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

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.

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.

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

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.

Molecular Enzymology of Phage T4 Dam DNA Methyltransferase

This review summarizes the results of a study of the molecular mechanisms of phage T4 DNA adenine methyltransferase (T4Dam) action. T4Dam [EC 2.1.1.72] catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to N6 of the adenine located in the palindromic recognition site GATC. The subunit structure of T4Dam, substrate-binding properties, and kinetic parameters of methylation of a variety of native and modified oligonucleotide duplexes are considered. A kinetic scheme of the reaction was proposed, assuming that T4Dam is isomerized into a catalytically active form. The mechanisms of DNA-induced dimerization of T4Dam, flipping of the target base, reorientation of T4Dam on an asymmetrically methylated recognition site, the effector action of substrates, and processive methylation of extended DNA containing more than one specific site are discussed. The results obtained for T4Dam may provide a better understanding of the action mechanisms of other homologous enzymes including, first and foremost, those of the vast Dam family.

Design of oligonucleotide inhibitors for human DNA methyltransferase 1

Mammalian DNA methyltransferase 1 (Dnmt1) is responsible for copying the DNA methylation pattern during cell division. Since Dnmt1 plays an important role in carcinogenesis, it is of particular interest to search for its specific inhibitors. To design oligonucleotide inhibitors of human Dnmt1, a number of singlestranded, double-stranded, and hairpin DNA structures containing a canonical or a modified Dnmt1 recognition site (5′-CG) were constructed on the basis of a 22-nt sequence. Structural features such as a C:A mismatch, phosphorothioates, and hairpins proved capable of incrementally increasing the oligonucleotide affinity for Dnmt1. An improvement of inhibitory properties was also achieved by replacing the target cytosine with 5,6-dihydro-5-azacytosine, 5-methyl-2-pyrimidinone, or 6-methyl-pyrrolo-[2,3-d]-2-pyrimidinone. The concentration that caused 50% inhibition of methylation of 1 μM poly(dI-dC) · poly(dI-dC), a conventional DNA substrate, was approximately 10−7 M for the most efficient oligonucleotides. Under the same in vitro conditions, these oligonucleotide inhibitors demonstrated a substantially stronger effect compared to known Dnmt1 inhibitors, which were used as controls.

Single Turnover Kinetics of Methylation by T4 DNA-(N6-Adenine)-Methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k1 = 0.21 s–1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k2 = 0.023 s–1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI MTase. Base substitutions and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme–substrate complexes is usually sharply reduced. The flipping of the adenosine residue to be modified in the recognition site upon interaction with the enzyme, revealed by fluorescence titration, supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.