Victor V. Zinoviev
State Research Center of Virology and Biotechnology VECTOR
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Journal of Biological Chemistry | 2003
Victor V. Zinoviev; Alexey A. Evdokimov; Ernst G. Malygin; Samuel L. Schlagman; Stanley Hattman
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.
Journal of Molecular Biology | 2003
William M. Lindstrom; Ernst G. Malygin; Lidiya G. Ovechkina; Victor V. Zinoviev; Norbert O. Reich
We show that the kinetic mechanism of the DNA (cytosine-N(4)-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGATCC), differs from cytosine C(5) methyltransferases, and is similar to that observed with adenine N(6) methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction. In contrast, the single-turnover rate of catalysis for M.BamHI (0.10s(-1)) is closer to the DNA (cytosine-C(5)-)-methyltransferases (0.14s(-1)) than the DNA (adenine-N(6)-)-methyltransferases (>200s(-1)). The nucleotide flipping transition dominates the single-turnover constant for adenine N(6) methyltransferases, and, since the disruption of the guanine-cytosine base-pair is essential for both types of cytosine DNA methyltransferases, this transition may be a common, rate-limiting step for methylation for these two enzyme subclasses. The similar overall rate of catalysis by M.BamHI and other DNA methyltransferases is consistent with a common rate-limiting catalytic step of product dissociation. Our analyses of M.BamHI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights.
Journal of Biological Chemistry | 2004
Ernst G. Malygin; Bianca Sclavi; Victor V. Zinoviev; Alexey A. Evdokimov; Stanley Hattman; Malcolm Buckle
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.
Molecular Biology | 2001
Ernst G. Malygin; Lidiya G. Ovechkina; Victor V. Zinoviev; U. M. Lindstrem; Norbert O. Reich
Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer duplex containing a palindromic recognition site GGATCC was studied by methods of steady-state and pre-steady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaraldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward the 20-mer substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower than the concentrations of substrates. The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s–1. The KM values for the duplex and SAM are 0.35 and 1.6 μM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s–1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in a single turnover of the enzyme, which coincides with the earlier data on EcoRI MTase. Regardless of the order of enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme–substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding with DNA. In gel retardation experiments, there was no stoichiometrically simple complex with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.
Molecular Biology | 2004
Victor V. Zinoviev; Alexey A. Evdokimov; Stanley Hattman; Ernst G. Malygin
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.
Molecular Biology | 2009
Alexey A. Evdokimov; Victor V. Zinoviev; V. V. Kuznetsov; N. A. Netesova; Ernst G. Malygin
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.
Archive | 2001
Ernst G. Malygin; Lidiya G. Ovechkina; Alexey A. Evdokimov; Victor V. Zinoviev
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.
Journal of Biological Chemistry | 2002
Alexey A. Evdokimov; Victor V. Zinoviev; Ernst G. Malygin; Samuel L. Schlagman; Stanley Hattman
Nucleic Acids Research | 1999
Ernst G. Malygin; Victor V. Zinoviev; Nicolay A. Petrov; Alexey A. Evdokimov; Linda Jen-Jacobson; Valeri G. Kossykh; Stanley Hattman
Journal of Biological Chemistry | 2003
Ernst G. Malygin; Victor V. Zinoviev; Alexey A. Evdokimov; William M. Lindstrom; Norbert. O. Reich; Stanley Hattman
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Dive into the Victor V. Zinoviev's collaboration.
State Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputsState Research Center of Virology and Biotechnology VECTOR
View shared research outputs