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)

The DNA of frog virus 3 (FV3), an iridovirus, is highly methylated; more than 20% of the cytosine bases are methylated at the 5-carbon position by an FV3-induced DNA methyltransferase (DNA-mt). To determine the role of this enzyme in virus replication and regulation of gene expression, we have analyzed an FV3 mutant that lacks DNA-mt activity and is resistant to 5-azacytidine (an inhibitor of DNA-mt). Comparative polypeptide analysis, using cytoplasmic extracts from the wild-type FV3 and mutant-infected cells, revealed that a single protein of 26,000 (26K) molecular weight was altered in the mutant-infected cells. The altered polypeptide migrated faster in SDS-polyacrylamide gel as compared to the wild-type FV3 26K protein. Five spontaneous revertants derived from the mutant regained the migrational characteristic of the wild-type 26K protein, DNA-mt activity, and methylation of their DNA. We further show that the 26K polypeptide is a DNA-binding protein and that 80% of the enzyme activity can be eluted from an ssDNA affinity column. Taken together, these data support the conclusion that the 26K polypeptide is associated with DNA-mt activity.
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PMID:Mutation in a DNA-binding protein reveals an association between DNA-methyltransferase activity and a 26,000-Da polypeptide in frog virus 3-infected cells. 244 2

The genome of the large icosahedral DNA virus, frog virus 3 (FV3), is heavily methylated at the cytosine residues of dCdG dinucleotide pairs, with more than 22% of the total cytosine residues in the form of 5-methylcytosine (5mC). This methylation is carried out postreplicatively in the cytoplasm of infected cells by a virus-encoded DNA methyltransferase. DNA methyltransferase activity was shown to copurify with a 26 kD virus-induced, DNA-binding protein that had an altered mobility in extracts from cells infected with a DNA-methyl-transferase deficient mutant of FV3. Immediately after infection, the highly methylated parental DNA is transcribed in the nucleus by the host cell RNA polymerase II. As FV3 induces the synthesis of a protein that can override the inhibitory effect of methylation on the transcription of exogenous promoters methylation in vitro, we suggest that this protein is a factor evolved by this virus to allow transcription from methylated promoters by eukaryotic RNA polymerase II.
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PMID:Transcription of methylated viral DNA by eukaryotic RNA polymerase II. 247 31

A nuclear protein isolated from human placenta, methylated DNA-binding protein (MDBP), binds selectively to DNA enriched in 5-methylcytosine. We now demonstrate that MDBP is a sequence-specific, as well as methylation-specific, DNA-binding protein. From ten restriction fragments of pBR322 DNA methylated with human DNA methyltransferase, one was bound to MDBP very much more strongly than any of the others. For this preferential binding to MDBP, the DNA had to be methylated. By a DNase I protection experiment (DNase I footprinting), a 22-base sequence within this methylated restriction fragment was shown to be specifically protected by MDBP. The sequence-specificity of MDBP coupled with its dependence on DNA methylation suggests that this is one of the proteins which modulates important functions of human DNA methylation in vivo.
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PMID:A human DNA-binding protein is methylation-specific and sequence-specific. 300 77

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.
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PMID:Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs. 302 66

The E2a gene of human adenovirus type 2 (Ad2) encodes the 72-kilodalton DNA-binding protein. We previously described perfect inverse correlations between the methylation of all 5' C-C-G-G 3' sequences in the Ad2 E2a gene in virus-transformed hamster cells containing viral DNA sequences in an integrated state and the extent to which this gene is expressed. We subsequently showed that in vitro methylation of all 14 5' C-C-G-G 3' sequences in the cloned E2a gene by prokaryotic Hpa II DNA methyltransferase leads to transcriptional inactivation after microinjection into Xenopus laevis oocytes. The unmethylated cloned E2a gene is expressed in these cells. We report here the construction of partly methylated clones of the E2a gene. In the promoter (5')-methylated construct, three 5' C-C-G-G 3' sequences at the 5' end of the subclone were methylated. One of these sites is located 215 base pairs (bp) upstream (bp 26,169 of Ad2 DNA), and two sites are located 5 and 23 bp downstream from the cap site (bp 25,931 and 25,949 of Ad2 DNA) of the E2a gene. This construct was transcriptionally inactive upon microinjection into nuclei of X. laevis oocytes. In the gene (3')-methylated construct, 11 5' C-C-G-G 3' sequences in the main part of the transcribed gene region were methylated in vitro. This construct was transcribed in X. laevis oocytes, and at least some of the Ad2-specific RNA synthesized was initiated at the same sites as in Ad2-infected human KB cells. Both mock-methylated constructs were transcribed into Ad2-specific RNA in X. laevis oocytes. These results demonstrate that DNA methylations at or close to the promoter and 5' end of the E2a gene cause transcriptional inactivation. Perhaps only one methyl group would be adequate for inactivation; in vivo methylation of more than one cytosine may be a form of safeguard or redundancy.
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PMID:DNA methylation of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2. 632 79

We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared. Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA.
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PMID:Human placental DNA methyltransferase: DNA substrate and DNA binding specificity. 653 66

In many viral and nonviral eukaryotic systems, an inverse correlation has been observed between the extent of DNA methylation at 5'-C-C-G-G-3' sites and the extent of expression of specific genes as mRNA. The E2a region of adenovirus serotype 2 (Ad2) DNA encodes the Ad2-specific DNA binding protein required for viral DNA replication. In three lines of Ad2 transformed hamster cells (HE1, HE2, and HE3), multiple copies of the major part of the Ad2 genome persist in an integrated state. Cell lines HE2 and HE3 do not express the DNA-binding protein whereas line HE1 does so. It has been shown that, in cell line HE1, all 5'-C-C-G-G-3' (Hpa II/MspI) sites in the E2a region remain unmethylated. Conversely, in lines HE2 and HE3 lacking expression of the E2a region all Hpa II sites are methylated. The cloned E2a region of Ad2 DNA, the HindIII A fragment in pBR322, was methylated in vitro by using Hpa II DNA methyltransferase (5'-C-C*G-G-3') or was left unmethylated. In vitro methylation did not break or nick supercoiled circular DNA. Methylated or unmethylated DNA was then microinjected into the nuclei of Xenopus laevis oocytes, and the subsequent synthesis of Ad2-specific RNA was monitored. In vitro-methylated DNA remained in the methylated state for 24 hr on microinjection into nuclei of xenopus oocytes; unmethylated DNA remained unmethylated. When the injected DNA had been methylated by using Hpa H DNA methyltransferase, Ad2-specific RNA was not synthesized as late as 24 hr after microinjection. Unmethylated DNA was readily expressed into Ad2-specific RNA. As an internal control, unmethylated histone genes (h22 DNA) from sea urchin were microinjected together with methylated E2a DNA from Ad2. Ad2-specific RNA was not found; h22 DNA-specific RNA was readily detected. This finding ruled out nonspecific inhibitory effects in the methylated DNA preparation. Ir was also shown that transcription of the unmethylated HindIII A fragment of Ad2 DNA in Xenopus oocytes was initiated on the late promoter of the E2a region. The same promoter was used in productively infected KB cells. Methylation by BsuRI methylse (5'-G'G-C*C-3') did not inactivate the HindIII A fragment. These results provide evidence for the notion that methylated sequences at highly specific sites are involved in the regulation of gene expression. The actual nature of the regulatory signal is not yet understood.
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PMID:Expression of a cloned adenovirus gene is inhibited by in vitro methylation. 695 Nov 63

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C. PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C. PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5' ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C. PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C. PvuII binding, overlapping the -10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.
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PMID:Role and mechanism of action of C. PvuII, a regulatory protein conserved among restriction-modification systems. 1062 96

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.
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PMID:The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine. 1267 53

We have previously reported that the gpt transgene in G12 Chinese hamster cells could be silenced by water-insoluble nickel compounds nickel sulfide (NiS) or nickel subsulfide (Ni(3)S(2)) and showed that the transgene was silenced by de novo DNA methylation and chromatin condensation. To further understand the nature of this silencing, we used the chromatin immunoprecipitation assay to elucidate the chromatin structure in nickel-induced silenced G12 clones. We also analyzed the effects of the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) and a histone deacetylase inhibitor trichostatin A (TSA) on histone H3 and H4 acetylation and gpt gene expression in selected nickel-silenced clones. We observed that both histone H3 and H4 were hypoacetylated and a methyl DNA-binding protein MeCP2 was targeted to the gpt gene locus, resulting in a localized inactive chromatin configuration in nickel-silenced cell clones. The histone H3K9 was also found methylated in three of four nickel- silenced cell clones, whereas the histone H3K9 was deacetylated in all four cell clones, indicating that the H3K9 methylation was involved in nickel-induced gene silencing. The acetylation of the gpt gene could be increased by a combination of 5-AzaC and TSA treatment, but not by either 5-AzaC or TSA alone. The gpt transcript was studied by either Northern blot or by semiquantitative RT-PCR following treatment of the silenced clones with TSA or 5-AzaC. An increase in gpt mRNA could be detected by RT-PCR in the clones that regained acetylation of H3 and H4. These data show that gene silencing induced by nickel in the gpt transgenic cell line involved a loss of histone acetylation and an activation of histone methylation. Both H4 and H3 histone acetylation were lost in the silenced clones and these clones exhibited an increase in the methylation of the lysine 9 in histone H3.
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PMID:Analysis of specific lysine histone H3 and H4 acetylation and methylation status in clones of cells with a gene silenced by nickel exposure. 1290 98


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