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)

O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.
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PMID:Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents. 165 27

Previously, we isolated and characterized six Bacillus subtilis ada mutants that were hypersensitive to methylnitroso compounds and deficient in the adaptive response to alkylation. Cloning of the DNA complementing the defects revealed the presence of an ada operon consisting of two tandem and partially overlapping genes, adaA and adaB. The two genes encoded proteins with methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase activities, respectively. To locate the six mutations, the ada operon was divided into five overlapping regions of about 350 bp. The fragments of each region were amplified by polymerase chain reaction and analyzed by gel electrophoresis to detect single-strand conformation polymorphism. Nucleotide sequences of the fragments exhibiting mobility shifts were determined. Three of the mutants carried sequence alterations in the adaA gene: the adaA1 and adaA2 mutants had a one-base deletion and insertion, respectively, and the adaA5 mutant had a substitution of two consecutive bases causing changes of two amino acid residues next to the presumptive alkyl-accepting Cys-85 residue. Three mutants carried sequence alterations in the adaB gene: the adaB3 mutant contained a rearrangement, the adaB6 mutant contained a base substitution causing a change of the presumptive alkyl-accepting Cys-141 to Tyr, and the adaB4 mutant contained a base substitution changing Leu-167 to Pro. The adaB mutants produced ada transcripts upon treatment with low doses of alkylating agents, whereas the adaA mutant did not. We conclude that the AdaA protein functions as the transcriptional activator of this operon, while the AdaB protein specializes in repair of alkylated residues in DNA.
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PMID:Molecular analysis of Bacillus subtilis ada mutants deficient in the adaptive response to simple alkylating agents. 174 39

cDNA for O6-methylguanine-DNA methyltransferase was isolated by screening rat liver cDNA libraries, using as a probe the human cDNA sequence for methyltransferase. The rat cDNA encodes a protein with 209 amino acid residues. The predicted amino acid sequence of the rat methyltransferase exhibits considerable homology with those of the human, yeast and bacterial enzymes, especially around putative methyl acceptor sites. When the cDNA was placed under control of the lac promoter and expressed in methyltransferase-deficient Escherichia coli (ada-, ogt-) cells, a characteristic methyltransferase protein was produced. The rat DNA methyltransferase thus expressed could complement the biological defects of the E. coli cell caused by lack of its own DNA methyltransferases; e.g. increased sensitivity to alkylating agents in terms of both cell death and mutation induction.
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PMID:Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase. 194 35

An efficient adaptive response to alkylation damage was observed in several enterobacterial species, including Klebsiella aerogenes, Shigella sonnei, Shigella boydii, Escherichia alkalescens, Escherichia hermanii, and Escherichia fergusonii. Increased O6-methylguanine-DNA and methylphosphotriester-DNA methyltransferase activities correlated with the induction of a 39-kDa protein recognized by monoclonal antibodies raised against the Escherichia coli Ada protein. Induced methyltransferase activities were similarly observed in Aerobacter aerogenes and Citrobacter intermedius, although no antigenically cross-reacting material was present. Weak induction of a 39-kDa protein immunologically related to the E. coli Ada protein occurred in Salmonella typhimurium. This protein encoded by the cloned S. typhimurium ada gene was shown to be an active methyltransferase which repaired O6-methylguanine and methylphosphotriesters in DNA as efficiently as did the E. coli Ada protein. However, the mehtyltransferase activity of the weakly induced 39-kDa protein in S. typhimurium was not detected, apparently because it was self-methylated and thus inactivated during the adaptive N-methyl-N-nitro-N-nitrosoguanidine pretreatment. In contrast, the E. coli ada gene on a low-copy-number plasmid was efficiently induced in S. typhimurium, and high methyltransferase activities were observed. We concluded that the inefficient induction of the adaptive response in S. typhimurium results from weak transcriptional activation of its ada gene by the self-methylated protein.
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PMID:A weak adaptive response to alkylation damage in Salmonella typhimurium. 205 Jun 26

The characteristics of O6-methylguanine-DNA methyltransferase (O6-MTase) produced in transgenic mice, in which the introduced E. coli ada gene was expressed under the control of the metallothionein promoter, were investigated. Liver extracts from transgenic homozygotes showed approximately 3 times the control activity, a marked increase of up to about 8 times the non-transgenic control levels being observed 10 h after zinc treatment. Examination of the substrate specificity of the enzyme revealed that the activity in the transgenic mice is due to the introduced foreign gene. The enzyme possessed methylphosphotriester-DNA methyltransferase as well as O6-MTase, characteristic of the E. coli Ada protein. Comparison of differences in biological response between transgenic and non-transgenic mice after treatment with the alkylating carcinogen methylnitrosourea (MNU) at various doses revealed transgenic mice to be more capable of repairing O6-MTase activity, only showing signs of exhaustion at very high levels of exposure. In non-transgenic mice, on the other hand, the basal level of O6-MTase was low, and the activity was hardly detectable when the animals were treated with MNU.
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PMID:Characterization of O6-methylguanine-DNA methyltransferase in transgenic mice introduced with the E. coli ada gene. 205 12

By prophage transformation and subcloning, we have obtained Bacillus subtilis DNA fragments that could complement the hypersensitivity of ada (adaptive response deficient) mutants to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The nucleotide sequence contained two open reading frames that were assigned to the genes adaA and adaB, encoding methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase, respectively. These two genes overlap by 11 bp and comprise a small operon. The 1.6 Kb transcripts derived from the operon were detected in ada+ cells cultured in the presence of MNNG but not in control ada+ cells. From analysis of the syntheses of DNA alkyltransferases in the ada mutant cells harboring the plasmid carrying the complete or partial fragment, we conclude that the adaA gene product functions as a transcriptional activator of the ada operon, while the adaB gene product specializes in repair of mutagenic O6-methylguanine residues. Comparison with Escherichia coli ada operon showed that the two genes correspond to portions of the E. coli ada gene, implicating gene fusion or splitting as the origin of the difference in the organizations of the genes.
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PMID:Bacillus subtilis ada operon encodes two DNA alkyltransferases. 212 Jun 77

To assess the biological role of DNA methylation at the O6 position of guanine (O6MeG) a human cell line was created that contains a regulatable gene of the O6MeG-DNA methyltransferase (MT), a repair activity that removes O6MeG adducts from the DNA. MT-deficient HeLa MR cells were transformed with an SV40-based expression vector in which the bacterial MT gene ada was put under the control of a glucocorticoid-inducible MMTV promoter. In response to dexamethasone (Dex), pSV MTV ada cells actively accumulated MT protein to reach a constant level after 10-12 h of approximately 15,000 MT molecules per cell. Co-induction by Dex and 12-O-tetradecanoylphorbol-13-acetate (TPA) further accelerated this synthesis approximately 2-fold and, as a result, higher final MT levels were achieved. The inducers were added to exponentially growing cells either before or at the time of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exposure and the kinetics of MT synthesis was studied. MNNG affected in a dose-dependent manner (i) the loss of the pre-existing MT activity; (ii) the lag before newly synthesized MT appeared; (iii) the final level of MT accumulated by the cells; and (iv) to a lesser extent the rate of MT synthesis. In cells with a down-regulated MT gene (no inducer) even small MNNG doses lead to an irreversible loss of the pre-existing MT activity, i.e. to incomplete repair, whereas an up-regulated MT gene supported the restoration of a pool of active MT molecules in the cells, i.e. an O6MeG repair that has gone to completion. Hence, effective O6MeG repair relies not only on the pre-existing MT level, but depends to an even greater extent on the state of expression of the MT gene. The activity of the MT gene also correlated with cell survival, which confirms our earlier finding that O6MeG adducts are cytotoxic for the cell.
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PMID:Effect of O6-methylguanine-DNA methyltransferase gene activity on repair in human cells transformed by a regulatable ada gene. 229 24

O6-Methylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O6-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction. The mammalian protein is highly regulated in both somatic and germ-line cells. In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein. The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada-) Escherichia coli host. A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E. coli methyltransferase (products of the ada and ogt genes), which encompass the alkyl-acceptor residues. The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E. coli genomic DNA. However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs. The lack of methyltransferase observed in many human cell lines is due to the absence of the MGMT gene or to lack of synthesis and/or stability of its 0.95-kilobase poly(A)+ RNA transcript.
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PMID:Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. 240 87

Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins. In mutant I the methyl acceptor Cys-321 for O6-methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted. Neither mutant protein had O6-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69. Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein. On the other hand, mutant II protein was inducible by N-methyl-N'-nitro-N-nitrosoguanidine, although to a smaller extent than the wild-type protein was, and the phosphotriester-DNA methyltransferase activity appeared to reside in 24- to 30-kilodalton cleavage products. Mutant I protein could be produced under lac promoter control, and its cleavage products, unlike those of mutant II protein, tended to aggregate. These results indicate that (i) Cys-321 cannot be replaced or transposed with the nucleophilic amino acid histidine for O6-methylguanine-DNA methyltransferase function, (ii) single amino acid replacement or transposition at the O6-methylguanine methyl acceptor site can have a profound effect on the in vivo stability and regulatory function of the Ada protein, and (iii) the integrity of the protein may not be absolutely needed for its transcription-activation function.
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PMID:Site-directed mutation of the Escherichia coli ada gene: effects of substitution of methyl acceptor cysteine-321 by histidine in Ada protein. 249 48

The E. coli ada gene encodes O6-methylguanine DNA methyltransferase (O6MTase) which repairs the methylation of guanine at the O6 position in DNA. After recombination with a Chinese hamster metallothionein I gene promoter, the ada gene was microinjected into C3H/HeN mouse zygotes. Eventually, transgenic mice containing the ada fusion DNA were generated. The integrated ada DNA complex was transmitted to the progeny in a mode conforming to tandem integration at a single chromosome site, and homozygotes were also obtained from an inter-transgenic mouse cross. RNA transcripts of the chimeric ada gene were identified in the livers of these transgenic mice using dot and Northern blot analyses. O6MTase activity was increased in the liver of transgenic mice of line No. 708, and was more than 3 times the activity found in non-transgenic mice, especially in the transgenic homozygotes. The ada gene product was detected in the liver of a transgenic homozygote by immunoblot analysis. These transgenic mice have great potential for analysis of the role played by O6MTase in chemical carcinogenesis.
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PMID:Enhanced O6-methylguanine-DNA methyltransferase activity in transgenic mice containing an integrated E. coli ada repair gene. 253 Apr 49


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