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 type IC DNA methyltransferase M.EcoR124I is a complex multisubunit enzyme that recognizes the non-palindromic DNA sequence GAAN6RTCG. Small angle X-ray scattering has been used to investigate the solution structure of the methyltransferase and of complexes of the enzyme with unmethylated and hemimethylated 30 bp DNA duplexes containing the specific recognition sequence. A major change in the quaternary structure of the enzyme is observed following DNA binding, based on a decrease in the radius of gyration from 56 to 40 A and a reduction in the maximum dimension of the enzyme from 180 to 112 A. The structural transition observed is independent of the methylation state of the DNA. CD shows that there is no change in the secondary structure of the protein subunits when DNA is bound. In contrast, there is a large increase in the CD signal arising from the DNA, suggesting considerable structural distortion which may allow access to the bases targeted for methylation. We propose that DNA binding induces a large rotation of the two HsdM subunits towards the DNA, mediated by hinge bending domains in the specificity subunit HsdS.
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PMID:DNA-binding induces a major structural transition in a type I methyltransferase. 798 73

Escherichia coli Dam DNA methyltransferase can methylate genomic GATC sites when expressed in Saccharomyces cerevisiae. Others have observed changes in the level of methylation at specific sites and suggested that these changes are related to transcriptional state or chromosomal context. To test directly the influence of nucleosome location on the ability of Dam methyltransferase to modify GATC sites in chromatin, we analyzed minichromosomes containing precisely positioned nucleosomes in dam-expressing yeast strains. Levels of methylation at individual GATC sites were rigorously quantified by an oligonucleotide-probing procedure. Within the linker and adjacent 21 bp of nucleosome-associated DNA, GATC sites were highly methylated, whereas methylation was severely inhibited by histone-DNA contacts nearer to the nucleosomal pseudodyad. Other DNA-protein complexes also interfere with Dam methylation. These data are consistent with a model in which nucleosomes exert a repressive influence on the biological functions of DNA by restricting access of trans-acting factors to DNA.
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PMID:Positioned nucleosomes inhibit Dam methylation in vivo. 810 16

O6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.
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PMID:Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening. 820 83

Caulobacter crescentus was found to have a DNA methyltransferase, CcrM, that methylates the adenine base of the HinfI recognition sequence, GANTC. The ccrM gene was cloned, and DNA sequence analysis revealed that the predicted amino acid sequence has 49% identity with the Haemophilus influenzae methyltransferase HinfM. Expression of the ccrM gene was found to be restricted to the portion of the cell cycle immediately prior to cell division. At three separate chromosomal sites the CcrM recognition sequence is fully methylated in swarmer cells, becomes hemimethylated upon DNA replication in stalked cells, and does not become remethylated until just prior to cell division. The time of methyltransferase expression coincides with the time of methylation of these three chromosomal sites and of plasmid DNA in the predivisional cell. When ccrM gene expression is placed under control of a constitutive promoter, these chromosomal sites are fully methylated throughout the cell cycle. A high proportion of morphologically aberrant cells, and cells that have undergone an additional chromosome replication initiation, are found in this population. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology.
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PMID:A Caulobacter DNA methyltransferase that functions only in the predivisional cell. 828 76

High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G. The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream. Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1. The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent.
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PMID:Complex transcription of an operon encoding the SalI restriction-modification system of Streptomyces albus G. 831 78

The first three-dimensional structure of a DNA methyltransferase is presented. The crystal structure of the DNA (cytosine-5)-methyltransferase, M.HhaI (recognition sequence: GCGC), complexed with S-adenosyl-L-methionine has been determined and refined at 2.5 A resolution. The core of the structure is dominated by sequence motifs conserved among all DNA (cytosine-5)-methyltransferases, and these are responsible for cofactor binding and methyltransferase function.
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PMID:Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine. 834 57

The DNA methyltransferase of the AluI restriction-modification system, from Arthrobacter luteus, converts cytosine to 5-methylcytosine in the sequence AGCT. The gene for this methyltransferase, aluIM, was cloned into Escherichia coli and sequenced. A 525-codon open reading frame was found, consistent with deletion evidence, and the deduced amino acid sequence revealed all ten conserved regions common to 5-methylcytosine methyltransferases. The aluIM sequence predicts a protein of M(r) 59.0k, in agreement with the observed M(r), making M.AluI the largest known methyltransferase from a type II restriction-modification system. M.AluI also contains the largest known variable region of any monospecific DNA methyltransferase, larger than that of most multispecific methyltransferases. In other DNA methyltransferases the variable region has been implicated as the sequence-specific target recognition domain. An in-frame deletion that removes a third of this putative target-recognition region leaves the Alu I methyltransferase still fully active.
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PMID:The M.AluI DNA-(cytosine C5)-methyltransferase has an unusually large, partially dispensable, variable region. 845 Nov 89

The cloning and overexpression of the MspI DNA methyltransferase as a functional fusion with glutathione S-transferase is described. The fusion enzyme retains full biological activity and has been used to investigate the interaction of substrates and inhibitors with MspI DNA methyltransferase. The fusion enzyme has been purified to homogeneity in a single step on GSH-agarose and is free from contaminating exonuclease activity. The enzyme can be photolabelled with S-adenosyl-L-methionine and the level of incorporation of label is enhanced by the presence of a nonspecific DNA duplex. In the presence of a cognate oligodeoxynucleotide, no photolabelling was observed since methyl transfer occurs instead. The inclusion of a mechanism-based inhibitor of C-5 deoxycytidine DNA methylation (an oligodeoxynucleotide containing the base 2-pyrimidinone-1-beta-D-2'-deoxyribofuranoside in the position of the deoxycytidine to which methyl addition occurs), which is thought to form a covalent interaction with the reactive cysteine of such enzymes, led to an enhancement of S-adenosyl-L-methionine photolabelling which suggests that, in contrast with results obtained with EcoRII DNA methyltransferase [Som and Friedman (1991) J. Biol. Chem. 266, 2937-2945], methylcysteine is not the photolabelled product. The implications of the results obtained with this mechanism-based inhibitor are discussed with respect to other C-5-specific DNA methyltransferases. Gel-retardation assays in the presence of cognate oligodeoxynucleotides that contain the reactive pyrimidinone base in place of the deoxycytidine target base are described. These demonstrate that most probably a stable covalent bond is formed between the methyltransferase and this oligodeoxynucleotide. However, the alternative of extremely tight non-covalent binding cannot be rigorously excluded. Furthermore, the results from these experiments indicate that the reaction mechanism proceeds in a manner similar to that of HhaI DNA methyltransferase with sequence-specific DNA binding being followed by addition of S-adenosyl-L-methionine and concomitant isomerization of the ternary complex leading to methyl transfer. S-Adenosyl-L-homocysteine appears to inhibit the reaction pathway as a result of either competition with the methyl donor and potentiation of a high-affinity interaction between the enzyme and DNA in an abortive ternary complex or through an allosteric interaction.
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PMID:Determination of the order of substrate addition to MspI DNA methyltransferase using a novel mechanism-based inhibitor. 848 30

High throughput chemical file screening with an enzymatic assay to detect inhibitors of the ErmC methyltransferase enzyme from macrolide-lincosamide-streptogramin B (MLSB) resistant pathogenic bacteria identified low molecular weight compounds that had IC50S (50% inhibitory concentration) in the nMolar to microMolar range. These same inhibitors were assessed in vitro for their capacity to inhibit the liver enzyme, cathechol-O-methyltransferase and the prokaryotic enzyme, EcoRI methylase. Selective inhibitors of the ErmC methyltransferase were tested in tertiary assays to determine their minimal inhibitory concentrations (MICs), as single agents and in combination with the macrolide, azithromycin, against strains of pathogenic bacteria expressing MLSB-resistance. Compounds that were active in vitro, alone or in combination with azithromycin, against strains of macrolide-resistant pathogens were tested in a mouse model of infection using an MLSB-resistant strain of Staphylococcus aureus or a macrolide-susceptible strain of Streptococcus pyogenes.
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PMID:Assays to detect and characterize synthetic agents that inhibit the ErmC methyltransferase. 855 68

In neoplastic cells, levels of DNA methyltransferase activity are often increased, and evidence is accruing to suggest an important role for this event in tumorigenesis. To evaluate this possibility further, and to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bacterial HhaI methyltransferase in cultured murine fibroblasts. This enzyme is a pure de novo DNA methyltransferase that methylates the internal C in the sequence GCGC. We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to the cells. However, surviving cell clones that express low levels of M. HhaI demonstrate increased tumorigenicity as assessed by soft agar cloning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0.4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 cells versus 0% for a mutant HhaI control) and tumorigenicity in nude mouse heterotransplants (75% for sense HhaI-transduced PA 317 cells versus 18.5% for antisense controls). DNA isolated from the clonogenic sense HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7-38.9) increase in methylcytosine content at GCGC sites. These findings suggest that eukaryotic cells tolerate a narrow window of increase de novo DNA methylating capacity, above which cell death occurs and within cell transformation results. Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.
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PMID:Expression of prokaryotic HhaI DNA methyltransferase is transforming and lethal to NIH 3T3 cells. 856 81


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