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)

DNA methylase has been purified 405-fold from Krebs II ascites cells. The purified enzyme is homogeneous on SDS-poly acrylamide gel electrophoresis (molecular weight about 80,000) and the only product of the reaction with DNA is 5-methyl cytosine. Both native and denatured DNA are methylated by the enzyme; with calf thymus DNA the double stranded form is the better substrate but the enzyme preferentially methylates single stranded E.coli DNA even in "native" preparations. Our results do not support a mechanism whereby the enzyme methylates DNA by binding irreversibly and "walking" along it. By measuring maximum levels of methylation of DNAs from different sources we have estimated the proportion of unmethylated sites present in them. Homologous ascites DNA can be methylated, but only to about 5% of the level of the best substrate, undermethylated mouse L929 cell DNA. DNA isolated from growing cells or tissues is a better substrate than DNA from normal liver or pancreas, or from stationary cells.
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PMID:DNA methylase: purification from ascites cells and the effect of various DNA substrates on its activity. 13 36

Histones (from calf thymus or from human placenta), if renatured in the presence of EDTA, caused a severe inhibition of in vitro methylation of double-stranded DNA (from Micrococcus luteus) by human placenta DNA methyltransferase. The absence of EDTA during the histone renaturation procedure abolished--at least in the 'physiological' range of the histones/DNA ratio--the inhibition. The H1 component was responsible for this inhibition, no effect being exerted by the other histones. H1 preparations were more effective if renatured in the presence of EDTA--90% inhibition being reached at a 0.3:1 (w/w) H1/DNA ratio. It seems likely that the requirement for the presence of EDTA during the renaturation process is correlated to its ability to induce a fairly stable ordered conformation of the histones, although this effect could also be shown with the 'inactive' H2a, H2b and H3 components, and was instead less evident with histone H1. The restriction to histone H1 of the ability to inhibit enzymic DNA methylation may account for the lower methylation levels present in the internucleosomal DNA of mammalian chromatin.
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PMID:Histones and DNA methylation in mammalian chromatin. Differential inhibition by histone H1. 188 42

Three molecular species of methyl-accepting proteins exist in Bacillus subtilis cells, which collect methyl groups from methylated DNA. A 20-kilodalton (kDa) protein was constitutively present in the cells of the ada+ (proficient in adaptive response) strain as well as in those of six ada (deficient in adaptive response) mutant strains and was assigned to the O6-methylguanine:DNA methyltransferase. Another species of O6-methylguanine:DNA methyltransferase, which had a molecular size of 22 kDa, emerged after adaptive treatment of the ada+ but not any of the ada mutant cells. A 27-kDa methyl-accepting protein, which preferred methylated poly(dT) to methylated calf thymus DNA as a substrate, was assigned to the methylphosphotriester:DNA methyltransferase. It was produced, after adaptive treatment, in the cells of ada+, ada-3, ada-4, and ada-6 strains but not in the cells of ada-1, ada-2, or ada-5 strains. These results support and extend our proposition that ada mutants can be classified into two groups; one (the ada-4 group) is defective only in the inducible synthesis of O6-methylguanine:DNA methyltransferase (22-kDa protein), and the other (the ada-1 group) is deficient in the adaptive response in toto. The finding that inducible and constitutive methyltransferases reside in different molecular species of methyl-accepting proteins is intriguing compared with the regulatory mechanisms of the adaptive response to simple alkylating agents in other organisms.
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PMID:Multiple species of Bacillus subtilis DNA alkyltransferase involved in the adaptive response to simple alkylating agents. 310 May 3

The effect of nutritional zinc-deficiency on the activities of O6-alkylguanine:DNA methyltransferase (AGT) in 9 rat tissues including liver, lung, kidney, spleen, brain, esophagus, forestomach, gastric-stomach and small intestine has been examined. Individual tissue extracts prepared from zinc-deficient and pair-fed, zinc-sufficient rats were incubated with N-[3H]methylnitrosourea-methylated calf thymus DNA for 1 h. The activities of AGT in these tissues were measured by two methods: (a) the transfer of the methyl group from O6-methylguanine in substrate DNA to AGT protein, and (b) the determination of the ratio of O6-methylguanine:7-methylguanine remaining in substrate DNA following incubation. AGT activities (expressed as fmol protein methylated/h per mg protein) were significantly reduced in the esophagus, spleen and lungs of zinc-deficient rats as compared to those in their corresponding zinc-sufficient counterparts. The ratio of O6-methylguanine:7-methylguanine was also reduced in the esophagus of the zinc-deficient rat. These results were consistent with our earlier findings that dietary zinc-deficiency enhances nitrosamine-induced esophageal carcinogenesis in rats.
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PMID:Effect of nutritional zinc-deficiency on O6-alkylguanine-DNA-methyl-transferase activities in rat tissues. 319 74

The effect of 3-alkyl substituted imidazotetrazinones on methylation of DNA has been studied in drug sensitive and resistant cell lines. The 3-methyl analogue (Temozolomide) has been shown to cause a decrease in the level of 5-methylcytosine in newly synthesized DNA in both cell lines, although the effect occurred at lower drug concentrations in the drug sensitive cell line. In order to investigate the mechanism of hypomethylation of DNA, calf thymus DNA was alkylated in vitro by both Temozolomide and the 3-ethyl analogue, CCRG 82019, and the alkylated DNA was shown to inhibit the transfer of methyl groups from S-adenosyl-L-methionine to M. lysodeikticus DNA by purified eukaryotic DNA methylase. Neither free drug alone or unmodified DNA affected the methylase reaction. Calf thymus DNA modified with CCRG 82019 was more effective as a methylase inhibitor than DNA modified with Temozolomide, which was a reverse of the order of potencies of the free drugs against tumour cells in culture. CCRG 82019 modified DNA also formed a more stable complex with nuclear proteins. Alterations in the level of 5-methylcytosine in DNA may be important in the alteration of gene expression by these agents.
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PMID:Antitumour imidazotetrazines and gene expression. 320 8

Two subpopulations of bovine calf thymus cells were separated by buoyant density centrifugation. The low-density cells (L-cells) showed high response to T-cell mitogens, while the high density cells (H-cells) did not. The DNA-metabolizing enzyme activities were elevated 10-fold in L-cells in comparison with those in H-cells. L-cells contained a DNA-replicating complex, DNA replitase, while H-cells did not. These observations suggested that L-cells were proliferating, mature T-cells and H-cells were dying intrathymic cells. A DNA methyltransferase was associated with DNA replitase in L-cells. In order to determine whether replitase-associated DNA methyltransferase functions at replicating regions, the methylation pattern of genomic DNA of L-cells was compared with that of H-cells. No significant difference was found in the extent of CpG dinucleotide methylation, and in the location of mC in the satellite I DNA sequence as identified by Southern hybridization and direct sequencing. Thus the majority of methylation patterns of genomic DNA did not change during T-cell development in the thymus. The results indicated that the methylation patterns were rapidly maintained in proliferating T-cells. Although methods employed in the present study might not be sensitive enough to detect transient hemimethylation, it is suggested that the rapid methylation might be catalyzed, albeit not completely, by a DNA methyltransferase associated with the DNA replitase complex.
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PMID:Rapid methylation of genomic DNA in proliferating T-cells from bovine thymocytes. 326 72

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

O6-Methylguanine-DNA methyltransferase, a DNA repair enzyme which transfers the methyl group of O6-methylguanine residue to a cysteinyl residue in the methyltransferase itself, was examined in rat organs by quantifying the S-methylcysteine formed in the methyl acceptor protein. Among the various organs examined, the spleen exhibited the highest enzyme specific activity followed by the thymus, liver, lung and testis. Brain had the lowest activity. The patterns of subcellular distribution of the methyltransferase in spleen and liver were different: while 75-80% of the activity was present in the nuclear fraction of the spleen, 54% of the activity in the liver was found in the nuclei and 35% in the cytosolic fraction. Forty-five and thirty-five percent of the total nuclear enzyme activity could be extracted with 1 M and 2 M NaCl solutions, respectively, indicating that the repair enzyme is not tightly bound to the nuclear matrix. When isolated nuclei were incubated with [methyl-3H]DNA substrate and subsequently fractionated into histone and non-histone protein fractions, over 90% of the radioactivity was coeluted on a Bio-Rex 70 column with the non-histone fraction and only a negligible amount of radioactivity was found to be associated with the histone fraction. The molecular mass of the [methyl-3H]methyltransferase in the non-histone fraction was determined to be 23,000, and its pI value was found to be 6.6 by two-dimensional polyacrylamide gel electrophoresis.
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PMID:Studies on the distribution of O6-methylguanine-DNA methyltransferase in rat. 394 77

A readily sedimentable nuclear fraction from Chinese hamster embryo fibroblast (CHEF/18) cells catalyzes incorporation of 14C-rCDP into DNA. Data indicated that this incorporation is made possible by the conversion of rCDP into a small and functionally compartmentalized, rather than a large and freely diffusible, pool of dCTP. This catalytically active sedimentable fraction from S phase CHEF/18 cells or actively replicating calf thymus cells contains nascent and template DNA, and numerous enzymes required for DNA biosynthesis including ribonucleoside diphosphate reductase, thymidylate synthetase, dihydrofolate reductase, DNA methylase, topoisomerase and DNA polymerase. We have named this catalytically active macromolecule the replitase. The replitase fraction contained spherical particles with a diameter of approximately 24 to 30 nm and had an estimated molecular weight on the order of 5 X 10(6).
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PMID:Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. 629 95

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


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