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)

Genes located near telomeres in Saccharomyces cerevisiae undergo position-effect variegation; their transcription is subject to reversible but mitotically heritable repression. This position effect and the finding that telomeric DNA is late replicating suggest that yeast telomeres exist in a heterochromatin-like state. Mutations in genes that suppress the telomeric position effect suggest that a special chromatin structure exists near chromosomal termini. Thus transcriptional repression may be explained by the inability of DNA binding proteins to access the DNA near telomeres. To test this hypothesis, the Escherichia coli Dam DNA methyltransferase, which modifies the sequence GATC, was introduced into S. cerevisiae cells. DNA sequences near the telomere were highly refractive to Dam methylation but were modified when located at positions more internal on the chromosome. Telomeric sequences were accessible to methyltransferase activity in strains that contained a mutation that suppressed the telomeric position effect. These data support the model that sequence-specific DNA binding proteins are excluded from telomere-proximal sequences in vivo and show that expression of DNA methyltransferase activity may serve as a useful tool for mapping chromosomal structural domains in vivo.
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PMID:Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. 157 Mar 34

The MspI restriction-modification system, which recognizes the sequence 5'-CCGG-3', has been previously cloned and sequenced (1). We subcloned the methyltransferase gene (M.MspI) downstream of the ptac promoter in the multicopy vector pUC119 and overexpressed it in E. coli. Upon induction with IPTG, M.MspI constitutes more than 10% of cellular protein. A scheme has been devised to purify large amounts of biologically active M.MspI to apparent homogeneity from these overexpressing E. coli cells. Approximately 0.8 mg of pure M.MspI per gram of cells (wet weight) can be obtained. The apparent molecular weight of M.MspI is 49 kD, by SDS gel electrophoresis and 48-54 kD by gel filtration. At low concentrations (less than 0.4 mg/ml), the methyltransferase is a monomer in solution but at higher concentrations (greater than 3.0 mg/ml) it exists predominantly as a dimer. Polyclonal antibodies raised against M.MspI cross-react with the DNA-methyltransferases of several other restriction-modification systems.
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PMID:Purification and characterization of the MspI DNA methyltransferase cloned and overexpressed in E. coli. 157 50

The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.
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PMID:Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation. 158 13

O6-Methylguanine-DNA methyltransferase plays an important role in preventing tumor induction. To elucidate the significance of a highly conserved amino acid sequence of methyltransferase protein, amino acid substitutions were introduced by site-directed mutagenesis of cloned cDNA for human methyltransferase and the activity and stability of mutant forms of enzyme were examined. When cysteine-145, to which the methyl transfer occurs, was replaced by other amino acids, all of the mutants isolated showed the methyltransferase-negative phenotype. From one of the negative mutants, methyltransferase-positive revertants were isolated, all of which carried codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and cannot be replaced by other amino acids. Using this negative and positive selection procedure, analyses were extended to other residues near the acceptor site. At the histidine-146 site, four substitutions (phenylalanine, methionine, asparagine and glutamine) exhibited the positive phenotype but the levels of methyltransferase activity in these mutants were low. With valine-148 substitutions there were six types of positive revertants, among which mutants carrying isoleucine, cysteine and alanine showed significantly high levels of methyltransferase activity. Some mutant forms of cDNA were expressed in methyltransferase-deficient human cells, and the results obtained with Escherichia coli cells were confirmed.
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PMID:Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity: analyses of three residues at or near the methyl acceptor site. 158 96

Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferase. This methyltransferase domain is linked to a large N-terminal domain. It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases. While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA. These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain. Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein. Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells.
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PMID:Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. 162 23

The sequence selectivity of enzyme-DNA interactions was analyzed by comparing discrimination between synthetic oligonucleotides containing the canonical site GAATTC and altered DNA sequences with the EcoRI DNA methyltransferase. The specificities (kcat/KmDNA) are decreased from 5- to 23,000-fold relative to the unmodified site. For several substrates the decrease in kcat makes a disproportionate contribution to the specificity difference, suggesting that discrimination is mediated by the placement of critical catalytic residues rather than binding interactions. This is supported by our observation that specificity changes are generally not followed by changes in the stability of the methyltransferase-DNA complexes. Also, base pair substitutions near the site of methylation result in greater decreases in complex stability, suggesting that recognition and catalytic mechanisms overlap.
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PMID:In vitro specificity of EcoRI DNA methyltransferase. 163 13

The dam gene of Escherichia coli encodes a DNA methyltransferase that methylates the N6 position of adenine in the sequence GATC. It was stably expressed from a shuttle vector in a repair- and recombination-proficient strain of Bacillus subtilis. In this strain the majority of plasmid DNA molecules was modified at dam sites whereas most chromosomal DNA remained unmethylated during exponential growth. During stationary phase the amount of unmethylated DNA increased, suggesting that methylated bases were being removed. An ultraviolet damage repair-deficient mutant (uvrB) contained highly methylated chromosomal and plasmid DNA. High levels of Dam methylation were detrimental to growth and viability of this mutant strain and some features of the SOS response were also induced. A mutant defective in the synthesis of adaptive DNA alkyltransferases and induction of the adaptive response (ada) also showed high methylation and properties similar to that of the dam gene expressing uvrB strain. When protein extracts from B. subtilis expressing the Dam methyltransferase or treated with N-methyl-N'-nitro-N-nitroso-guanidine were incubated with [3H]-labelled Dam methylated DNA, the methyl label was bound to two proteins of 14 and 9 kD. Some free N6-methyladenine was also detected in the supernatant of the incubation mixture. We propose that N6-methyladenine residues are excised by proteins involved in both excision (uvrB) and the adaptive response (ada) DNA repair pathways in B. subtilis.
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PMID:Expression of Escherichia coli dam gene in Bacillus subtilis provokes DNA damage response: N6-methyladenine is removed by two repair pathways. 164 27

Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II restriction endonuclease, FsuI, which recognizes the sequence 5'-GG(A/T)CC-3'. Analysis of the restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 5' protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II restriction-modification system of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium.
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PMID:Type II DNA restriction-modification system and an endonuclease from the ruminal bacterium Fibrobacter succinogenes S85. 164 54

Mammalian DNA cytosine-5-methyltransferase (MTase, EC 2.1.1.37) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome. The cDNA for the murine enzyme was previously cloned in segments. We have reconstructed the entire gene, encoding a protein of 1517 amino acids, from a set of overlapping cDNA clones. We report the assembly of two expression constructs in bacterial/mammalian shuttle vectors. Transcription in the first construct (pEMT) is driven by the cytomegalovirus enhancer/promoter and encodes a fusion protein with 15 additional aa at the N terminus, while the second construct (pJMT) is driven by the simian virus 40 early promoter/enhancer upstream from the natural ATG codon. Immunofluorescence microscopy and immunoblot analysis have shown that both constructs direct the synthesis of MTase in COS-1 cells. Enzyme activity in whole-cell lysates of transfected COS-1 cells transfected with pEMT and pJMT are on average tenfold and fivefold higher than in controls, respectively. The specific activities of the recombinant and endogenous mouse-cell enzyme are similar. These expression constructs will be of use in studies of DNA methylation in mammals.
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PMID:Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase. 166 57

We initiated this study to determine whether three structurally related bifunctional alkylating agents could induce the expression of a presumptive human DNA repair gene. The gene chosen for this study is known to encode the ribosomal phosphoprotein PO, but ironically may also share functions related to DNA repair. We now show by Northern analysis that PO is induced by L-phenylalanine mustard, 4-hydroperoxycyclophosphamide and mechlorethamine, which are DNA-damaging agents commonly used as chemotherapeutic antitumor agents. In further support of its involvement in DNA repair is the finding of a 30- to 50-fold constitutive overexpression of the PO gene in human tumor cell lines that are Mer-, cells which lack O6-methylguanine methyltransferase activity, when compared to Mer+ cell lines. This constitutively elevated level of PO in Mer- cell lines, which are thus DNA repair defective for O6-alkyguanine lesions, was not observed for other genes tested, including the human ribosomal gene S17 whose mRNA steady-state levels were uniformly the same in both Mer- and Mer+ cells. Taking these data together, it appears that increased levels of PO are somehow linked to DNA repair, and increased expression of PO may compensate for the decreased O6-methylguanine DNA methyltransferase activity in Mer- cells. Furthermore, the PO gene has also been shown to be overexpressed in colorectal tumors and polyps and the sera of some systemic lupus erythematosus patients contain antibodies against PO. The titer of the anti-PO antibodies rises significantly during lupus psychosis.
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PMID:Expression of ribosomal phosphoprotein PO is induced by antitumor agents and increased in Mer- human tumor cell lines. 174 17


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