Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.1.1.37 (DNA methyltransferase)
 4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The origin and function of the large amount of 5-methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300-fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE-cellulose, Sephadex G-75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in double-stranded DNAs isolated from wheat, maize, calf thymus or bacteria using S-adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de novo and in maintenance methylation. A relative molecular mass of 50,000-55,000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of Mr = 50,000 and one other component (Mr = 35,000). The preference for endogenous, double-stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA.
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PMID:DNA methylation in wheat. Purification and properties of DNA methyltransferase. 362 12

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.
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PMID:Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA. 367 27

We investigated the methylation reaction catalyzed by 1500-fold purified rat liver DNA methyltransferase (DMase) on native Micrococcal luteus DNA (ML-DNA) and poly(dC-dG) templates containing covalently bound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the strongly carcinogenic, principal metabolite of benzo[a]pyrene. Since eukaryotic DNA methyltransferases recognize the dinucleotide 5'd[CG] in DNA as a substrate for methylation, the model polynucleotide poly(dC-dG) was used to study in more detail the mode of interaction and effect on incorporation. With either of these BPDE-modified templates, a progressive inhibition of methylation was correlated with increasing amount of BPDE substitution. The effect of BPDE-dG adducts did not alter the apparent km with respect to the concentration of d[CG] in either unmodified or BPDE-modified poly(dC-dG) (km = 10 microM) but lowered the relative apparent Vmax. In assays in which perturbation by salt of preformed enzyme-DNA complex is measured, no change in the relative stability to either unsubstituted or the carcinogen-modified template was noted, thus, excluding any change in the ionic component of this interaction. However, in competition-type experiments, BPDE-DNA is an inhibitor of the methylation reaction on native DNA. When BPDE-DNA is allowed to interact with the enzyme before the addition of native competitor DNA, the methylation rate is not stimulated, suggesting very tight hydrophobic binding of the enzyme to BPDE-DNA and an inhibition in the dissociation of DMase from the template following a methylation event.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of rat liver DNA methyltransferase interaction with anti-benzo[a]pyrenediol epoxide modified DNA templates. 609 97

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
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PMID:Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. 658 75

DNA methyltransferase activity, present in low salt extracts of nuclei from young pea shoot apices, has been fractionated into two different species by assaying with model substrates. The CG methyltransferase (an unstable enzyme believed to be of 140 kDa) methylates cytosine only in oligonucleotides with CG and Cl dinucleotide targets while an enzyme of 110 kDa (the CNG methyltransferase) methylates the cytosines in 5'-CAG-3' and 5'-CTG-3' target sequences, especially when hemimethylated, but not in 5'-CCG-3' nor in 5'-CGG-3' target sequences present in oligonucleotides.
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PMID:Distinct CG and CNG DNA methyltransferases in Pisum sativum. 775 18

DNA methylase, present in low-salt extracts of nuclei prepared from Pisum sativum shoot tips, methylates model DNA substrates containing CNG trinucleotides or CI dinucleotides only. The binding to the hemimethylated trinucleotide substrates is very much stronger and more persistent than the binding to the unmethylated substrates or to the hemimethylated dinucleotide substrate. When the DNA concentration is limiting, the rate of methyl-group transfer with the hemimethylated CNG substrate is much greater than that with the unmethylated CNG. However, the Vmax. is similar for the two CNG substrates. On fractionation using Q-Sepharose, two peaks of activity are seen with different relative activities using the di- and trinucleotide substrates. The relative activity with these substrates changes during purification, during plant growth and on heating at 35 degrees C as well, indicating that more than one enzyme or more than one form of the enzyme may be present.
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PMID:DNA substrate specificity of pea DNA methylase. 835 29

DNA methylase activity of brain in Wistar rats treated with Shenfang Bushen Shengxue Drug (SBSD) was observed using 3H-labelled methyl group of S-adenosyl-methionine incorporated into DNA. It was found that the SBSD has a marked effect on anti-aging. After SBSD treatment the specific activity of DNA methylase increased, the thermostability and salt-tolerance of it improved slightly, its optional pH changed from 7.5 to 8.0. A and a difference was found between the electrophoresgram of partially purified product of DNA methylase in SBSD treated rats and that in normal rats, suggesting SBSD could change the activity and characteristics of DNA methylase so as to affect the level of DNA methylation, which might be one pathway of SBSD effects on anti-aging.
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PMID:[Effect of anti-aging drug on DNA methylase of brain in rats]. 938 66

The EcoRV DNA methyltransferase (M.EcoRV) specifically methylates the first adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly influenced by the length of oligonucleotide substrates employed. If substrates >20 bp compared to a 12mer substrate, the kcat/Km increases 100-fold, although the enzyme does not contact more than 12 base-pairs on the DNA. Single-turnover rates are higher than kcat 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-specific substrates (d ln(KAss)/d ln(cNaCl) = - 2, indicative of electrostatic interactions) and specific substrates (d ln(KAss)/d ln(cNaCl) = + 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 pKa 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 flipping out the target adenine cannot contribute to the rate-limiting step of the enzymatic reaction.
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PMID:Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase. 948 Jul 66

The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation of Caulobacter crescentus have been elucidated by using defined unmethylated or hemimethylated DNA (DNAHM) substrates. Catalytic efficiency is significantly enhanced with a DNAHM substrate. Biphasic kinetic behavior during methyl incorporation is observed when unmethylated or DNAHM substrates are used, indicating that a step after chemistry limits enzyme turnover and is most likely the release of enzyme from methylated DNA product. The enzyme is thermally inactivated at 30 degrees C within 20 min; this process is substantially decreased in the presence of saturating concentrations of DNAHM, suggesting that the enzyme preferentially binds DNA before S-adenosylmethionine. The activity of the enzyme shows an unusual sensitivity to salt levels, apparently dissociating more rapidly from methylated DNA product as the salt level is decreased. The enzyme acts processively during methylation of specific DNA sequences, indicating a preferred order of product release in which S-adenosylhomocysteine is released from enzyme before fully methylated DNA. The kinetic behavior and activity of the enzyme are consistent with the temporal constraints during the cell cycle-regulated methylation of newly replicated chromosomal DNA.
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PMID:A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA. 950 Nov 83

Cyanothece sp. ATCC 51142 possesses six host restriction-modification systems (HRMS), and these HRMS were designated as Csp68KI to Csp68KVI. So far, four restriction enzymes have been characterized biochemically, and the other two were deduced based on the cloning of corresponding DNA methyltransferase genes M. Csp68KIV and M. Csp68KV from the Cyanothece genome. Csp68KI, Csp68KII, Csp68KIII, Csp68KIV, Csp68KV, and Csp68KVI are isoschizomers of AvaII, AsuII, AvaIII, MspI, HaeIII, and FnuDII, respectively. The cleavage specificities for Csp68KI, Csp68KII, Csp68KIII, and Csp68KVI were characterized. The Cyanothece restriction enzymes showed different temperature and salt requirements for their optimal activity. For example, the restriction enzyme Csp68KII functions optimally at 50 degreesC and Csp68KIII requires higher salt for its activity.
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PMID:Restriction-modification systems in the marine, aerobic, nitrogen-fixing unicellular cyanobacterium Cyanothece sp. ATCC 51142. 970 42


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