Purification and biochemical characterization of the Ecal DNA methyltransferase (original) (raw)
Related papers
Gene, 1995
Two additional conserved motifs (CM), CM Is and CM III, have been found in addition to well-known CM I and CM II within the primary amino acid sequences of almost all m6A-and m4C-methyltransferases (MTases). The boundaries of all four CM were defined and their consensus sequences characteristic both for different classes, as well as for all N-MTases, were derived. Some regular deviations at fixed positions of the consensus sequences CM Is, CM I and CM II, typical for separate classes of N-MTases, were presumed to correlate. A possible structural basis for the supposed interregional correlations is discussed and experiments for verification of the assumed interactions between CM are suggested. A classification scheme for all N-MTases is provided.
Kinetic characterization of the EcaI methyltransferase
European Journal of Biochemistry, 1993
A kinetic analysis of the EcaI adenine-N6-specific methyltransferase (MTase) is presented. The enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the adenine of the GGTNACC sequence with a random rapid-equilibrium mechanism. Experiments with a synthetic, 14-bp DNA substrate suggest that recognition of the specific site of DNA occurs after the binding of AdoMet. Proton concentration does not affect the dissociation constant of AdoMet while V,, and the dissociation constant of DNA show a maximum around pH 8. Increasing the amount of S-adenosyl-L-homocysteine decreases the inhibitory effect of methylated DNA which proves the active role of AdoMet in site recognition. Experiments with hemimethylated DNA show that the methylase binds the double-stranded DNA asymmetrically.
The non-specific adenine DNA methyltransferase M.EcoGII
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...
The EcoRV DNA-(adenine-N 6 )-methyltransferase recognizes GATATC sequences and modi®es the ®rst adenine residue within this site. We show here, that the enzyme binds to the DNA and the cofactor S-adenosylmethionine (AdoMet) in an ordered bi-bi fashion, with AdoMet being bound ®rst. M.EcoRV binds DNA in a non-speci®c manner and the enzyme searches for its recognition site by linear diffusion with a range of approximately 1800 bp. During linear diffusion the enzyme continuously scans the DNA for the presence of recognition sites. Upon speci®c M.EcoRV-DNA complex formation a strong increase in the¯uorescence of an oligonucleotide containing a 2-aminopurine base analogue at the GAT-2AP-TC position is observed which, most likely, is correlated with DNA bending. In contrast to the GAT-2AP-TC substrate, a G-2AP-TATC substrate in which the target base is replaced by 2-aminopurine does not show an increase in¯uorescence upon M.EcoRV binding, demonstrating that 2-aminopurine is not a general tool to detect base¯ipping. Stoppedow experiments show that DNA bending is a fast process with rate constants >10 s À1 . In the presence of cofactor, the speci®c complex adopts a second conformation, in which the target sequence is more tightly contacted by the enzyme. M.EcoRV exists in an open and in a closed state that are in slow equilibrium. Closing the open state is a slow process (rate constant %0.7 min À1 ) that limits the rate of DNA methylation under single turnover conditions. Product release requires opening of the closed complex which is very slow (rate constant %0.05-0.1 min À1 ) and limits the rate of DNA methylation under multiple turnover conditions. M.EcoRV methylates DNA sequences containing more than one recognition sites in a distributive manner. Since the dissociation rate from non-speci®c DNA does not depend on the length of the DNA fragment, DNA dissociation does not preferentially occur at the ends of the DNA.
Nucleic Acids Research, 1989
The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacterfreundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.QC9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.
Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase
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.
DNA binding and methyl transfer catalysed by mouse DNA methyltransferase
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.
Influence of Pre-existing Methylation on the de Novo Activity of Eukaryotic DNA Methyltransferase †
Biochemistry, 1998
Aberrant de novo methylation of CpG island DNA sequences has been observed in cultured cell lines or upon malignant transformation, but the mechanisms underlying this phenomenon are poorly understood. Using eukaryotic DNA (cytosine-5)-methyltransferase (of both human and murine origin), we have studied the in vitro methylation pattern of three CpG islands. Such sequences are intrinsically poor substrates of the enzyme, yet are efficiently methylated when a small amount of 5-methylcytosine is randomly introduced by the M.SssI prokaryotic DNA (cytosine-5)-methyltransferase prior to in vitro methylation by the eukaryotic enzyme. A stimulation was also found with several other double-stranded DNA substrates, either natural or of synthetic origin, such as poly(dG-dC)‚poly(dG-dC). An A+T-rich plasmid, pHb 1S, showed an initial stimulation, followed by a severe inhibition of the activity of DNA (cytosine-5)-methyltransferase. Methylation of poly(dI-dC)‚poly(dI-dC) was instead inhibited by preexisting 5-methylcytosines. The extent of stimulation observed with poly(dG-dC)‚poly(dG-dC) depends on both the number and the distribution of the 5-methylcytosine residues, which probably must not be too closely spaced for the stimulatory effect to be exerted. The activity of the M.SssI prokaryotic DNA methyltransferase was not stimulated, but was inhibited by pre-methylation on either poly(dG-dC)‚poly-(dG-dC) or poly(dI-dC)‚poly(dI-dC). The prokaryotic and eukaryotic DNA methyltransferases also differed in sensitivity to poly(dG-m 5 dC)‚poly(dG-m 5 dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.