Kinetic characterization of the EcaI methyltransferase

Nucleic acids research, 2017

We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo - using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using puri...

Journal of Molecular Biology, 1997

the active site amino acid residues 105 NPPY 108. Dissociation constants for the Molekulare Physiologie complexes of M.TaqI with the three ligands were determined spec-Abteilung Physikalische trofluorometrically. Sinefungin binds more strongly than S-adenosyl-L-ho-Biochemie, Rheinlanddamm mocysteine or S-adenosyl-L-methionine, with K D =0.34 mM, 2.4 mM and 201, D-44139 Dortmund 2.0 mM, respectively. Germany

The EcoRV DNA methyltransferase (M.EcoRV) speci®cally methylates the ®rst adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly in¯uenced by the length of oligonucleotide substrates employed. If substrates >20 bp compared to a 12mer substrate, the k cat /K m increases 100-fold, although the enzyme does not contact more than 12 base-pairs on the DNA. Single-turnover rates are higher than k cat values. M.EcoRV binding to DNA is fast but dissociation from the DNA is slow, demonstrating that the multiple-turnover rate is limited by the rate of product release. The kinetics of DNA binding by M.EcoRV are not in accordance with the thermodynamics binding constant, suggesting that the M.EcoRV-DNA complex is involved in a slow conformational change. The salt dependence of DNA binding is different for non-speci®c substrates (d ln(K Ass )/d ln(c NaCl ) À 2, indicative of electrostatic interactions) and speci®c substrates (d ln(K Ass )/d ln(c NaCl ) 1, indicative of hydrophobic interactions). This result demonstrates that the M.EcoRV-DNA complex has a different conformation in both binding modes. M.EcoRV does not discriminate between hemimethylated and unmethylated substrates. Using the 20mer we have analyzed the temperature and pH dependence of the single-turnover rate constant of M.EcoRV-DNA methylation by M.EcoRV has an activation energy of 40 kJ/mol and its rate increases with increasing pH. The pH dependence reveals the presence of an ionizable residue with a pK a of 7.9, which must be unprotonated for catalysis. The rates of DNA methylation remain unchanged if an abasic site is introduced instead of the thymidine residue that is base-paired to the target adenine, demonstrating that ipping out the target adenine cannot contribute to the rate-limiting step of the enzymatic reaction.

The Biochemical journal, 1995

By using a purified fraction of mouse DNA methyltransferase we have shown, by gel-retardation analysis, that the enzyme forms a low-affinity complex preferentially with hemimethylated DNA; the complexes formed with unmethylated or with fully methylated DNA are of even lower affinity, and only very weak interaction occurs with DNA lacking CG dinucleotides. Interaction is inhibited by N-ethylmaleimide. Methyl transfer from S-adenosyl-methionine is associated with the release of the fully methylated product from the complex. Complexes formed with the intact enzyme are extremely large, but limited trypsin treatment allows a major complex to enter the gel. DNA binding is not inhibited by this limited proteolysis of the native enzyme.

Proceedings of the National Academy of Sciences, 1994

The Thermus aualcus DNA methyltransferase M'Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of-CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethlonlne shows a/P folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide deft suitable to accommodate the DNA substrate. The N-terminal domain Is dominated by a nine-stranded 3-sheet; it contains the two conserved segments typical for N-methyltranserases which form a pocket for cofactor binding. The C-terminal domain is formed by four small 1-sheets and a-helices. The three-dimensional folding of M'Taq I is similar to that of the cytosine-speciflc Hha I methyltransferase, where the large 1-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.

Journal of Biological Chemistry, 2001

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-L-methionine (AdoMet) and product S-adenosyl-L-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI⅐AdoMet complex (k off ‫؍‬ 22 s ؊1 , K D ‫؍‬ 6 M) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI⅐ Me DNA⅐AdoHcy (k chem ‫؍‬ 0.26 s ؊1 ) is followed by a slower decay step (k off ‫؍‬ 0.045 s ؊1 ), which is the rate-limiting step in the catalytic cycle (k cat ‫؍‬ 0.04 s ؊1 ). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K D DNA(ternary) , k off DNA(ternary) , K M AdoMet(ternary) values but small changes in K D DNA(binary) , K D AdoMet(binary) , k chem , and k cat . When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI⅐DNA complex is inhibited 400fold (k chem 5FC ‫؍‬ 0.7 ؋ 10 ؊3 s ؊1 ), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k chem . We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k chem ) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.

Nucleic Acids Research, 1990

An enzymatic activity rendering DNA Immune to the action of the S/nal restriction endonuclease in the presence of S-adenosyl-L-methionine has been detected in Serratla marcescens Sb. This methylase, M.S/nal, modifies the second cytosine residue of the substrate sequence CCCGGG yielding N4-methylcytoslne.

Journal of Molecular Biology, 2003

Co-transfections of reporter plasmids and plasmids encoding the catalytic domain of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene expression. As Dnmt3a mutants with C ! A and E ! A exchanges in the conserved PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, we checked for their catalytic activity in vitro. Surprisingly, the activity of the cysteine variant and of the corresponding full-length Dnmt3a variant is only two to sixfold reduced with respect to wild-type Dnmt3a. In contrast, enzyme variants carrying E ! A, E ! D or E ! Q exchanges of the ENV glutamate are catalytically almost inactive, demonstrating that this residue has a central function in catalysis. Since the glutamic acid residue contacts the flipped base, its main function could be to hold the target base at a position that supports methyl group transfer. Whereas wild-type Dnmt3a and the ENV variants form covalent complexes with 5-fluorocytidine modified DNA, the PCN variant does not. Therefore, covalent complex formation is not essential in the reaction mechanism of Dnmt3a. We propose that correct positioning of the flipped base and the cofactor and binding to the transition state of methyl group transfer are the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl group transfer.