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 ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1

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

O6-Methylguanine DNA methyltransferase (MGMT; EC 2.1.1.63) is an unusual DNA repair protein in that it directly and specifically repairs a premutagenic DNA lesion without involving other proteins. MGMT removes the alkyl group from O6-alkylguanine in DNA in a unique stoichiometric reaction by accepting the alkyl group on a cysteine residue. The intracellular level of MGMT varies among tissues and appears to be inversely correlated to tissue-specific tumorigenesis induced by monofunctional alkylating agents. Because MGMT acts in solo, genetic manipulation of its expression may provide valuable insight into its contribution to cellular resistance to alkylation toxicity and to tumor induction. The human MGMT full length cDNA has been fused with a portion of the human transferrin (TF) 5'-flanking region (TF/MGMT). Transgenic founder mice were produced carrying the TF/MGMT transgene and then bred to establish stable transgenic lines. Human MGMT transcripts were specifically expressed in abundance in transgenic brain and liver tissues. In vitro MGMT assays revealed approximately 150-fold and approximately 25-fold increases in MGMT activity in transgenic brain and liver extracts respectively. Western blot analysis confirmed that human MGMT protein is specifically synthesized in transgenic brain and liver tissues.
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PMID:Brain and liver targeted overexpression of O6-methylguanine DNA methyltransferase in transgenic mice. 835 38

The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG). The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site. One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases. The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E. coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4 degrees C and 37 degrees C. The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37 degrees C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4 degrees C. Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures. Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold. We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.
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PMID:The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity. 836 18

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

A 0.5 kb fragment of chicken DNA methyltransferase cDNA was PCR-amplified using a set of degenerate primers. A clone harboring a 5 kb insert was isolated from a cDNA library by screening with the PCR-amplified cDNA fragment as a probe. The elucidated nucleotide sequence gave a 4,614 nucleotide open reading frame, and the predicted protein was highly homologous to the mouse and human DNA methyltransferases, especially in the amino acid sequence of the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and Lys-Gly repeat first found in the mouse sequence were also conserved in chicken. However, about 250 amino acid residues in the amino-terminal portion of chicken DNA methyltransferase diverged from the amino-terminus of the mouse or human sequence. Northern blot analysis showed that the message of chicken DNA methyltransferase was expressed at high levels in the testis, in the lung and in Marek's virus-transformed chicken T-lymphoma cells. Expression of the chicken DNA methyltransferase in COS1 cells demonstrated that the enzyme is a so-called maintenance-type methylase. When poly(dG-dC)-poly(dG-dC) was used as the methyl acceptor, to provide a measure of de novo methylase activity, the Km value for S-adenosyl L-methionine was about 5 microM, which was 10 times higher than that when poly(dI-dC)-poly(dI-dC) was used. The affinity of DNA methyltransferase for S-adenosyl L-methionine in catalyzing de novo-type methylation activity was lower than that in catalyzing maintenance-type activity, though it was still high enough for the enzyme to work as a de novo-type methylase under physiological conditions.
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PMID:Isolation and expression of a chicken DNA methyltransferase cDNA. 858 18

O6-Methylguanine-DNA methyltransferase (MGMT) is strongly involved in drug resistance mechanism of tumor cells to chloroethylnitrosoureas (CENUs), because it removes and repairs CENU-induced O6-alkylguanine-DNA by accepting the alkyl group at a cysteine moiety. MGMT activity and MGMT mRNA expression are good indicators for detection of sensitive cells or resistant cells to CENUs. In the present study, we applied a non-radioactive reverse transcription-polymerase chain reaction (RT-PCR) method on quantitative measurement of MGMT mRNA expression. Estimated levels of MGMT mRNA expression determined by this RT-PCR method were consistent with the actual doses of MGMT mRNA. This relationship was noted at a wide range from 10 fg to 10 pg. The relative expression levels of MGMT mRNA estimated from kinetic analysis correlated well with MGMT activity determined using 3H-methyl-nitrosourea-treated DNA substrate in brain tumor cells (P<0.001 with a correlation coefficient of 0.997). The RT-PCR method facilitated quantitative measurements in even a small amount of biopsy specimens obtained by stereotactic brain surgery.
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PMID:Quantification of O6-methylguanine-DNA methyltransferase mRNA in human brain tumors. 860 18

Cysteine residue 69 of the Escherichia coli Ada transcription factor, which accepts a methyl group from methylphosphotriester in methylated DNA, was substituted by each of 19 other amino acids. Only the mutant Ada (C69H), carrying a histidine substitution of Cys69, exhibited a limited degree of transactivating potential for the ada promoter in E. coli cells although the mutant protein was completely devoid of methylphosphotriester-DNA methyltransferase activity. Using a multicopy plasmid system for the expression of Ada protein, we have shown that Ada C69H has a transactivating capacity equivalent to that of wild-type Ada protein in the absence of an alkylating agent. This indicates that the zinc-binding capacity of histidine at residue 69 is likely to be sufficient for Ada to recognize and bind to the ada promoter. Furthermore, transactivation of the ada promoter by Ada C69H was enhanced up to 6-fold by treatment with methylating agents. An additional substitution was made with alanine in Ada C69H, replacing Cys321, the site for acceptance of a methyl group from O6-methylguanine and O4-methylthymine residues in DNA, with alanine. This renders the protein completely inactive as a methyltransferase but this derivative is constitutively active as a transactivator for the ada promoter. Therefore, acquisition of a methyl group at Cys321 apparently enhances the transactivating capacity of Ada protein on the ada promoter. We propose that the transcription-regulating function of Ada protein is under dual control by methylation of cysteine residues at positions 69 and 321; the former enhances DNA binding, while the latter enhances the transactivating capacity of the protein.
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PMID:Requirement for two conserved cysteine residues in the Ada protein of Escherichia coli for transactivation of the ada promoter. 867 55

O6-methylguanine DNA methyltransferase (MGMT) is a repair protein that transfers methyl groups from O6-methylguanine to a cysteine acceptor in its own molecule, and restores DNA to its undamaged state. If left unrepaired, O6-methylguanine can pair with either a thymine or a cytosine, causing a C-G to T-A transition, which is considered to be one of the molecular mechanisms of both mutagenesis and carcinogenesis. The expression of MGMT mRNA in liver tissue was quantitatively assessed by the competitive reverse transcription-polymerase chain reaction method in patients with chronic liver diseases with or without alcohol drinking. MGMT mRNA expression was 1.4 +/- 0.9 pg/micrograms RNA in control livers. Its expression in chronic hepatitis was 3.8 +/- 0.7 in alcoholics and 2.7 +/- 0.8 in nonalcoholics, which were not statistically different. MGMT mRNA expression in liver cirrhosis was significantly low, compared with that in chronic hepatitis, and 0.8 +/- 0.3 in alcoholics and 0.5 +/- 0.1 in nonalcoholics, which also were not significantly different. The present study shows that MGMT mRNA was not decreased in patients with chronic liver diseases with alcohol drinking, compared with those without alcohol drinking.
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PMID:Effect of alcohol drinking on gene expression of hepatic O6-methylguanine DNA methyltransferase in chronic liver diseases. 898 26

A Xenopus DNA methyltransferase cDNA was isolated from a Xenopus oocyte cDNA library by screening with the mouse DNA methyltransferase cDNA as a probe. The elucidated nucleotide sequence gave a 4,470 nucleotide open reading frame, and the predicted protein was composed of 1,490 amino acid residues, showing high homology to animal DNA methyltransferases, especially in the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and the Lys-Gly repeat which were first found in the mouse sequence were conserved in Xenopus. However, 200 amino acid residues at the amino-terminus of Xenopus DNA methyltransferase were quite different from those of mouse and human, but showed 70% homology with those of chicken. The cloned Xenopus DNA methyltransferase cDNA expressed in COS1 cells showed a significant DNA methyltransferase activity. The size of the translation product of Xenopus DNA methyltransferase cDNA expressed in COS1 cells was identical with that of the endogenous DNA methyltransferase in Xenopus A6 cells and also with the size of newly synthesized DNA methyltransferase in Xenopus oocytes. However, a slightly larger immunoreactive band of about 205 kDa, and a small immunoreactive band of about 100 kDa, which were poorly labeled by short incubation with radiolabeled amino acids, were the main bands in stage I-III and stage IV-VI oocytes, respectively.
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PMID:Isolation and expression of a Xenopus laevis DNA methyltransferase cDNA. 901 Jul 68


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