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)

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.
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PMID:Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism. 1856 10

Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 degrees C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 degrees C. rMcrA protein, which is predicted to contain a Cys(4)-Zn(2+) finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high alpha-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm(5)CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in "restricting" m(5)C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m(5)C residues.
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PMID:Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease. 1866 88

Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA(Asp-GTC), tRNA(Val-AAC) and tRNA(Gly-GCC) are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.
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PMID:RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage. 2067 93

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, Dictyostelium discoideum and Drosophila. Our recent work has shown that Ehmeth methylates tRNA(Asp), and this finding indicates that this enzyme has a dual DNA/tRNA(Asp) methyltransferase activity. This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.
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PMID:In vitro tRNA methylation assay with the Entamoeba histolytica DNA and tRNA methyltransferase Dnmt2 (Ehmeth) enzyme. 2104 66

Drosophila belongs to the so-called "Dnmt2 only" organisms, and does not contain any of the canonical DNA methyltransferases (Dnmt1 and Dnmt3). Furthermore, no functional homologs of known 5-methylcytosine reader proteins are found. Nevertheless, there is strong evidence for DNA methylation in this organism. It has been suggested that DNA methylation in Drosophila is simply a byproduct of Dnmt2, which is a DNA methyltransferase (Dnmt) according to structure and type of catalysis but functions in vivo as a tRNA methyltransferase. However, concerning the very specific timing of cytosine methylation in Drosophila, their suggested functions in control of retrotransposon silencing and genome stability, and the obvious DNA methylation activity of Dnmt2 enzymes in the protozoans Dictyostelium discoideum and Entamoeba histolytica, we tend to disagree with this notation. Dnmt2 probably serves, and not only in Drosophila, as a methyltransferase of both specific DNA and tRNA targets.
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PMID:DNA methylation in Drosophila--a critical evaluation. 2150 51

The Dnmt2 enzymes show strong amino acid sequence similarity with eukaryotic and prokaryotic DNA-(cytosine C5)-methyltransferases. Yet, Dnmt2 enzymes from several species were shown to methylate tRNA-Asp and had been proposed that eukaryotic DNA methyltransferases evolved from a Dnmt2-like tRNA methyltransferase ancestor [Goll et al., 2006, Science, 311, 395-8]. It was the aim of this study to investigate if this hypothesis could be supported by evidence from sequence alignments. We present phylogenetic analyses based on sequence alignments of the methyltransferase catalytic domains of more than 2300 eukaryotic and prokaryotic DNA-(cytosine C5)-methyltransferases and analyzed the distribution of DNA methyltransferases in eukaryotic species. The Dnmt2 homologues were reliably identified by an additional conserved CFT motif next to motif IX. All DNA methyltransferases and Dnmt2 enzymes were clearly separated from other RNA-(cytosine-C5)-methyltransferases. Our sequence alignments and phylogenetic analyses indicate that the last universal eukaryotic ancestor contained at least one member of the Dnmt1, Dnmt2 and Dnmt3 families of enzymes and additional RNA methyltransferases. The similarity of Dnmt2 enzymes with DNA methyltransferases and absence of similarity with RNA methyltransferases combined with their strong RNA methylation activity suggest that the ancestor of Dnmt2 was a DNA methyltransferase and an early Dnmt2 enzyme changed its substrate preference to tRNA. There is no phylogenetic evidence that Dnmt2 was the precursor of eukaryotic Dnmts. Most likely, the eukaryotic Dnmt1 and Dnmt3 families of DNA methyltransferases had an independent origin in the prokaryotic DNA methyltransferase sequence space.
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PMID:On the evolutionary origin of eukaryotic DNA methyltransferases and Dnmt2. 2214 May 15

The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNA(Asp) and, to a lesser extent, of tRNA(Glu), despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.
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PMID:Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling. 2307 92

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.
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PMID:Dnmt2-dependent methylomes lack defined DNA methylation patterns. 2364 Oct 3

Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.
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PMID:Comprehensive DNA methylation analysis of the Aedes aegypti genome. 2780 64

The amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNA^Asp, we showed that this natural Dnmt2 substrate could be engineered to employ RNAs that act like guide RNAs in vitro. The 5'-half of tRNA^Asp was able to efficiently guide methylation toward a single stranded tRNA fragment as would result from tRNA cleavage by tRNA specific nucleases. In a more artificial setting, a composite system of guide RNAs could ultimately be engineered to enable the enzyme to perform cytidine methylation on single stranded DNA in vitro.
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PMID:The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA. 2781 23


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