Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.
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PMID:Inhibition of EcoRI DNA methylase with cofactor analogs. 234 14

DNA in mammalian cells is enzymatically methylated at the 5-position of cytosine via S-adenosylmethionine and DNA methyltransferase. Several chemical carcinogens have been shown to inhibit this reaction, altering DNA methylation. We have been studying the mechanism by which carcinogens alter the methylation of DNA in order to better understand the cellular regulation of DNA methylase activity and to understand the role, if any, of DNA methylation in the carcinogenic process. We have utilized an in vitro assay for DNA methylase isolated from purified rat-liver nuclei. Ethionine, a liver carcinogen, given to rats 17 hr after partial hepatectomy inhibited the incorporation of [methyl-3H]-methionine into 5-methylcytosine residues of DNA. DNA isolated from these ethionine-treated rats was able to accept methyl groups from S-adenosylmethionine 8 times more than control DNA. It was further demonstrated that S-adenosylethionine competitively inhibited the DNA methylase resulting in hypomethylated DNA. N-Methyl-N-nitro-N-nitrosoguanidine reacted with the DNA methylase at the sulfhydryl sites inactivating the enzyme. Methylnitrosourea did not react directly with the methylase enzyme, but when reacted with DNA, the DNA methylase activity was inhibited by the carcinogen alkylated DNA. Sodium selenite also inhibited the enzyme non-competitively with a Ki of 6.7 microM. 5-Azacytidine prevented the 2 to 3 fold increase in DNA methylase seen 2 days following partial hepatectomy. All of these data with various carcinogens, altering DNA methylation by different mechanisms, support the hypothesis that DNA methylation plays a role in the initiation of carcinogenesis.
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PMID:Studies on DNA methyltransferase and alteration of the enzyme activity by chemical carcinogens. 243 29

DNA containing 5-azacytidine (5-azaC) has been shown to form stable detergent-resistant complexes with cytosine methylases. We reasoned that if 5-azaC treatment causes protein-DNA cross-links in vivo, then mutations in DNA repair and recombination genes may increase the sensitivity of a cell to 5-azaC. We found that although recA (defective) and lexA (induction-negative) mutants of Escherichia coli were very sensitive to the drug, mutations in uvrA and ung genes had little effect on cell lethality. The sensitivity of recA strains to 5-azaC was dose dependent and was enhanced by the overproduction of a DNA cytosine methylase in the cell. Unexpectedly, a strain of E. coli carrying a recA mutation and a deletion of the DNA cytosine methylase gene (dcm) was found to be significantly sensitive to 5-azaC. Study of mutations in the pyrimidine salvage pathway of E. coli suggests that direct phosphorylation of 5-azaC, rather than phosphorylation of its degradation products, is largely responsible for the lethal effects of the drug. The addition of uracil to the growth medium has little effect on cell lethality of recA mutants, but it partially reversed the inhibition of cell growth caused by 5-azaC. This reversal of the bacteriostatic effects of the drug could not be achieved by adding cytosine or orotic acid to the growth medium and required the presence of functional UMP-pyrophosphorylase (gene upp) in the cell.
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PMID:Genetic analysis of the 5-azacytidine sensitivity of Escherichia coli K-12. 243 6

Survival and mutagenesis caused by 5-azacytidine was studied in Escherichia coli. Survival was partially lexA- and recA-dependent and was decreased by the presence of a DNA (cytosine-5)methyltransferase. The dcm, MspI, and EcoRII methyltransferase genes all decreased survival. There was no direct relationship between amount of methylase enzyme present and cell survival, but only plasmids containing a methylase gene sensitized cells to 5-azacytidine. Survival was not affected by uvrA, uvrB or umuCD mutations. Induction of sulA::lacZ fusions by 5-azacytidine was inhibited in strains containing elevated levels of DNA methylase. Cells resistant to 5-azacytidine when they contained a plasmid specifying the EcoRII methylase were sensitive if the plasmid specified the complete EcoRII restriction-modification system. The mechanism of cell death in these situations is therefore different. Mutation of the rpoB gene by 5-azacytidine was studied. The mutation rate was decreased by the presence of recA and lexA mutations. Mutation in umuCD had little effect on the mutation rate. The recA430 mutation, which does not support SOS-dependent mutagenesis induced by UV light, does support 5-azacytidine induced mutagenesis. The presence of DNA (cytosine-5)methyltransferase had no effect on the mutation rate caused by 5-azacytidine treatment. The mutagenic and lethal lesions caused by 5-azacytidine in the absence of methylase therefore differ from the lethal lesions that occur in the presence of methylase. The former could be due to the opening of the 5-azacytosine ring in DNA. Cell death in the presence of methylase could be due to tight binding of methylase to azacytosine containing DNA as well as inhibition of induction of the SOS response.
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PMID:Survival and mutagenic effects of 5-azacytidine in Escherichia coli. 245 47

DNA methylase extracted with low salt from mouse Krebs II ascites cell nuclei has been degraded stepwise by trypsin treatment. Degradation, accompanied by a limited reduction in size of the native enzyme, leads to the progressive introduction of several nicks so that, eventually, fragments of 14, 18, 24 and 28 kD are released on denaturation. This illustrates the domain structure of the enzyme. In contrast to ascites cell nuclear extracts, preparations from liver nuclei are already nicked and the major from of the enzyme contains a 100 kD fragment though the native molecular weight is unchanged. Newborn mouse liver contains more undegraded enzyme that is mostly firmly-bound within the nucleus. Trypsin treatment increases the de novo activity of the enzyme and prevents its aggregation in the absence of salt, even in the presence of high concentrations of native DNA.
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PMID:Mouse DNA methylase. Intracellular location and degradation. 247 19

The advantages of assaying of DNA methylase by measuring the transfer to water of tritium from the 5 position of DNA cytosine, rather than the transfer to DNA of labeled methyl groups are discussed.
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PMID:Measurement of DNA methylase activity by tritium release from DNA cytosine. 247 21

Upon extensive digestion with DNAaseI of placenta chromatin matrix, previously "stripped" from its loosely-bound components by high-salt extraction, a fraction is obtained that contains almost no endogenous DNA methylase activity but whose DNA, if still included in this whole fraction--not if it has been purified to a protein-free condition--is a good substrate for externally added enzyme. This chromatin matrix can even cause a significant stimulation of methylation of single-stranded Micrococcus luteus DNA by placental methylase. In vivo, this phenomenon may have possible counterparts in the existence of highly-methylated regions of chromatin loops that appear to be protected by tightly-bound protein components from digestion of the "stripped loops" with DNAaseI.
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PMID:A possible role of chromatin and tightly-bound chromatin proteins on enzyme-catalyzed methylation of DNA. 247 22

The methylation status of the 160 kD viral capsid antigen (VCA) gene promoter was determined by hybridization analysis. The semi-permissive marmoset cell line FF41-1 lacked cytosine methylation in approximately three quarters of the VCA promoter CpG dinucleotide residues. In the stringently infected HH514CL16 cell line the same CpG residues were methylated in three quarters of the genomes. 5'deoxy-5'-S-isobutyladenosine (SIBA), a DNA methylase inhibitor, was utilized to disrupt the EBV latent state. As determined by flow cytometry, SIBA treatment significantly increased expression of VCA. The VCA promoter was hypomethylated in VCA-positive FF41-1 cells sorted by flow cytometry. While hypomethylation alone was not sufficient for VCA transcriptional activity, the absence of methylation of VCA promoter CpG dinucleotide residues was associated with expression of VCA.
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PMID:Relationship between methylation status and expression of an Epstein-Barr virus (EBV) capsid antigen gene. 253 21

DNA cytosine methylation and transcription of specific genes are inversely correlated. In granulocytic differentiation of HL-60 cells there is a distinct down regulation of the c-myc proto-oncogene expression, which is probably a causal mechanism. With differentiation of HL-60 cells we found no restriction enzyme fragment length polymorphism (RFLP) within the c-myc proto-oncogene, which indicates that there is no loss of regulatory elements (e.g., TATAA boxes within the first exon). Furthermore, we found no de novo methylation in this region. Methylation of other DNA regions, which could influence c-myc expression, is also not necessary for differentiation, as was shown by inhibition of DNA methylase. L-Ethionine and S-adenosyl-L-homocysteine are both potent inhibitors of DNA methylase and do not influence proliferation of HL-60 cells, as shown by FACS analysis.
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PMID:Granulocytic differentiation of HL-60 cells is not regulated by DNA de novo methylation. 256 92

It is shown that the methylation of DNA that occurs in isolated nuclei is "delayed methylation". This methylation is not reduced in nuclei which have been pretreated with 0.2M NaCl to extract the soluble methylase suggesting that this methylation is the product of a firmly bound matrix associated DNA methylase. Evidence is provided that, like the methylase, the DNA substrate is associated with the nuclear matrix.
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PMID:Delayed methylation and the matrix bound DNA methylase. 257 43


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