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 (O6-MT) has been described as a DNA repair enzyme that reverses alkylation damage at the O6 position of guanine in DNA. We demonstrate that the concentration of this protein decreases immediately prior to DNA synthesis in cultured chick hepatocytes. If intracellular levels are experimentally depleted by treatment of cultures with O6-methylguanine, DNA synthesis occurs as an associated resultant. This effect is dose dependent and can be followed by discernible morphological changes of organoids in culture. Increased and altered growth caused by O6-methylguanine was quantified and was also found to be dose dependent. Therefore, O6-MT may play a role in the regulation of DNA synthesis.
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PMID:Relationship between the depletion of O6-methylguanine-DNA methyltransferase by O6-methylguanine and the stimulation of DNA synthesis and growth of cultured chick hepatocytes. 173 70

As a step toward the molecular elucidation of the putative replicational apparatus associated with the nuclear matrix, we have investigated the possible matrix association of several replicational related enzymes. In addition to the previously identified DNA polymerase alpha, DNA primase, 3'-5' exonuclease, RNase H, and DNA methylase were all recovered at significant levels (20-30% of total nuclear activity) in nuclear matrix isolated from regenerating rat liver during maximal in vivo replication (22 h post-hepatectomy). In contrast, DNA ligase was not detected on the nuclear matrix even though significant activity was present in isolated nuclei. Examination of the replicative dependency of these enzyme activities following partial hepatectomy revealed pre-replicative elevations which were distinct for each matrix-bound enzyme. A second late-replicative peak in DNA methylase is consistent with a role of this matrix-bound enzyme in the maintenance of the inheritable methylation pattern. Mild sonication resulted in a significant release of all of these activities except RNase H. A major portion of the matrix-solubilized DNA polymerase alpha, DNA primase, 3'-5' exonuclease, and DNA methylase activities cosedimented on sucrose gradients between approximately 8-12 S. Our results are consistent with the organization of at least a portion of these replicative enzymes into nuclear matrix-bound replicational complexes. We also propose a novel pre-replicative assembly model of the matrix-bound replicational apparatus in which DNA primase plays an initial and critical role.
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PMID:Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration. 302 82

O6-Methylguanine-DNA methyltransferase, a DNA repair enzyme which transfers the methyl group of O6-methylguanine residue to a cysteinyl residue in the methyltransferase itself, was examined in rat organs by quantifying the S-methylcysteine formed in the methyl acceptor protein. Among the various organs examined, the spleen exhibited the highest enzyme specific activity followed by the thymus, liver, lung and testis. Brain had the lowest activity. The patterns of subcellular distribution of the methyltransferase in spleen and liver were different: while 75-80% of the activity was present in the nuclear fraction of the spleen, 54% of the activity in the liver was found in the nuclei and 35% in the cytosolic fraction. Forty-five and thirty-five percent of the total nuclear enzyme activity could be extracted with 1 M and 2 M NaCl solutions, respectively, indicating that the repair enzyme is not tightly bound to the nuclear matrix. When isolated nuclei were incubated with [methyl-3H]DNA substrate and subsequently fractionated into histone and non-histone protein fractions, over 90% of the radioactivity was coeluted on a Bio-Rex 70 column with the non-histone fraction and only a negligible amount of radioactivity was found to be associated with the histone fraction. The molecular mass of the [methyl-3H]methyltransferase in the non-histone fraction was determined to be 23,000, and its pI value was found to be 6.6 by two-dimensional polyacrylamide gel electrophoresis.
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PMID:Studies on the distribution of O6-methylguanine-DNA methyltransferase in rat. 394 77

A rapid assay of O6-MeG-DNA methyltransferase activity is described. Following incubation of cell extracts with O6-[3H]MeG-containing DNA, remaining radioactive DNA was hydrolyzed in trichloroacetic acid and separated from methylated radioactive protein by filtration or centrifugation. Transfer of radioactive methyl from DNA to protein was proportional to the amount of protein added, and was not linear with time. More than 90% of the radioactivity precipitated after acid hydrolyses was in S-methyl cysteine residues. The method was used to measure O6-MeG-DNA methyltransferase activity in extracts of 24 neoplastic tissues from human organs. Although five tumor tissues had 28-84% lower activity of O6-MeG-DNA methyltransferase than the corresponding normal tissue from the same patient, higher or similar levels of activity were found more frequently. Thus, a lack of O6-MeG-DNA methyltransferase activity in human tumours appears not to be a frequent event. The DNA repair enzyme uracil-DNA glycosylase was also measured in the same extracts. Most frequently the level of uracil-DNA glycosylase activity was essentially similar in tumors and normal tissues but significantly higher or lower levels were also observed.
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PMID:A simplified assay for O6-methylguanine-DNA methyltransferase activity and its application to human neoplastic and non-neoplastic tissues. 674 14

O6-Benzylguanine (O6BG) enhances the cytotoxicity of the nitrosoureas by irreversibly binding and inhibiting the DNA repair enzyme O6-methyl-guanine-DNA methyltransferase (MGMT). The plasma and cerebrospinal fluid (CSF) pharmacokinetics of O6BG and its active metabolite, O6-benzyl-8-oxoguanine, were studied in a nonhuman primate model after 200 mg/m2 had been injected i.v. The parent drug and the metabolite were measured with a reverse-phase HPLC assay. A pharmacokinetic model incorporating separate compartments for O6BG and the O6-benzyl-8-oxoguanine metabolite, first-order conversion of O6BG to the metabolite, and additional first-order elimination rate constants for each compound, was simultaneously fitted to the parent drug and metabolite plasma concentration time data. Elimination of O6BG from plasma was rapid; it had a half-life of 1.6 h and a clearance of 68 ml/min/m2. On the basis of the pharmacokinetic model, essentially all of the O6BG was converted to O6-benzyl-8-oxoguanine. The plasma pharmacokinetic profile of the metabolite differed considerably from that the parent drug. The half-life (14 h) was 10-fold longer and the area under the curve (2420 microM/h) was 11-fold higher than that of O6BG (212 microM/h). The clearance rate of O6-benzyl-8-oxoguanine was 6.4 ml/min/m2. The CSF:plasma ratio was 4.3% for O6BG and 36% for O6-benzyl-8-oxoguanine, and the metabolite area under the curve was 90-fold higher than that of O6BG in CSF. The excellent CSF penetration of the active metabolite provides a rationale for the use of O6BG as a chemosensitizing agent for brain tumors. We also studied the duration of MGMT inhibition in peripheral blood mononuclear cells. By 2 h after a 200 mg/m2 dose of O6BG, > 98% of MGMT activity was suppressed, and > 95% suppression of enzyme activity persisted at 18 and 48 h after the dose. By 2 weeks after the treatment, MGMT levels had returned to baseline. Persistent high concentrations of the active metabolite appear to provide a pharmacological explanation for the prolonged suppression of MGMT activity.
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PMID:Plasma and cerebrospinal fluid pharmacokinetics of O6-benzylguanine and time course of peripheral blood mononuclear cell O6-methylguanine-DNA methyltransferase inhibition in the nonhuman primate. 755 37

Oxidation of DNA and its components can cause genetic mutations and chromosomal instability. These changes have generally been implicated in aging. Oxidation of the methyl group of thymidine residues in DNA is known to result in the formation 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd). We have utilized Bacillus subtilis phage SPO1 DNA as a model of oxidatively damaged DNA. In this phage, all thymine (Thy) residues are replaced by 5-hydroxymethyluracil (5HmUra), but the species is naturally devoid of other oxidatively-induced DNA lesions. Particular attention was paid to the behavior of 5HmUra-containing DNA as a target for several enzymes employing DNA as substrate; restriction endonucleases, dam DNA methylase and T4 DNA ligase. We noticed that susceptibility of SPO1 DNA varied when different restriction endonucleases having 5HmUra in the restriction sites were tested. Endonucleolytic cleavage brought about Sau3A proceeded as effectively with SPO1 DNA as with conventional DNA (lambda phage). The same was true when the ligation of Sau3A sites was performed with T4 DNA ligase. In contrast, both endonucleolytic cleavage and ligation were slower in SPO1 DNA, compared with lambda phage, when Taq I and T4 DNA ligase were used for restriction and ligation, respectively. We also noticed that SPO1 phage does not naturally contain N6-methyladenine (N6MeAde) opposite 5HmUra, i.e., no hydrolysis of SPO1 DNA was observed when assessed with methylation-dependent restriction endonuclease DpnI. Our results show that the presence of 5HmUra in the respective site of DNA does not, per se, prevent the activity of restriction endonucleases, ligases or DNA methylases. These data support the view that oxidation of Thy to 5HmUra in target DNA does not necessarily result in substantial deterioration in the functions of DNA processing enzymes.
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PMID:Restriction, methylation and ligation of 5-hydroxymethyluracil-containing DNA. 786 75

The DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT) is responsible for repair of damage induced by alkylating agents that produce adducts at O6-guanine in DNA. Although the MGMT gene promoter has housekeeping gene promoter characteristics, unlike these genes which are expressed at a constant level, MGMT transcriptional activity varies between cell types. During an attempt to identify regions of the MGMT regulatory sequence sensitive to variations in transcription factors between cell types, we have identified a 59 bp enhancer which is required for efficient MGMT promoter function. This fragment produced increased transcriptional activity in reporter gene constructs containing either the MGMT or UMP-synthase promoter when transfected into either of two cell lines; it seems therefore that this enhancer may interact with relatively common trans-acting factors. Functional activity is only detected when the enhancer is in 'cis' with respect to the promoter, suggesting that complexes are formed between proteins bound to the enhancer and promoter sequences. We propose that the enhancer-binding protein may be a novel transcription factor since there are no obvious consensus sequences within the 59 bp sequence for known DNA-binding proteins.
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PMID:Identification of a 59 bp enhancer located at the first exon/intron boundary of the human O6-methylguanine DNA methyltransferase gene. 798 9

O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer+) and -deficient (Mer-) cells, correlated with the endogenous MGMT activity in Mer+ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5'-CTGGGTCGC-3') for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer+ cells tested but was apparently restricted to the cytoplasm of Mer- cells. We conclude that the MEBP-enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer+ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer- cells.
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PMID:Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells. 911 92

This report completes a preliminary analysis of the sequence of the 330,740-bp chlorella virus PBCV-1 genome, the largest virus genome to be sequenced to date. The PBCV-1 genome is 57% the size of the genome from the smallest self-replicating organism, Mycoplasma genitalium. Analysis of 74 kb of newly sequenced DNA, from the right terminus of the PBCV-1 genome, revealed 153 open reading frames (ORFs) of 65 codons or longer. Eighty-five of these ORFs, which are evenly distributed on both strands of the DNA, were considered major ORFs. Fifty-nine of the major ORFs were separated by less than 100 bp. The largest intergenic distance was 729 bp, which occurred between two ORFs located in the 2.2-kb inverted terminal repeat region of the PBCV-1 genome. Twenty-seven of the 85 major ORFs resemble proteins in databases, including the large subunit of ribonucleotide diphosphate reductase, ATP-dependent DNA ligase, type II DNA topoisomerase, a helicase, histidine decarboxylase, dCMP deaminase, dUTP pyrophosphatase, proliferating cell nuclear antigen, a transposase, fungal translation elongation factor 3 (EF-3), UDP glucose dehydrogenase, a protein kinase, and an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease. Seventeen of the 153 ORFs resembled other PBCV-1 ORFs, suggesting that they represent either gene duplications or gene families.
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PMID:Analysis of 74 kb of DNA located at the right end of the 330-kb chlorella virus PBCV-1 genome. 935 47

A recent study reported that exposure of student embalmers in Cincinnati to high concentrations of formaldehyde (2 mg/m3) reduced the activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Reduction in a DNA repair enzyme may strongly increase the cancer risk not only with respect to the repair-enzyme causing agent but with respect to all carcinogens causing lesions subject to repair by the enzyme in question. Thus, we examined whether formaldehyde exposure of 57 medical students during their anatomy course at two different Universities in Germany influenced MGMT activity in mononuclear blood cells. Mean formaldehyde exposure of 41 students was 0.2 +/- 0.05 mg/m3 for 6 h per week. MGMT activity was 133.2 +/- 14.9 fmol MGMT/10(6) cells before the beginning of the formaldehyde exposure, 131.1 +/- 15.8 fmol MGMT/10(6) cells after 50 days (P = 0.56) and 128.2 +/- 19.0 fmol MGMT/10(6) cells after 111 days of exposure (P = 0.92). Similarly, no significant influence of formaldehyde exposure was observed, when smoking habits, alcohol consumption, allergic disease and sex of students were considered. In addition no significant difference was obtained in MGMT activity between 16 students with mean formaldehyde exposure of 0.8 +/- 0.6 mg/m3 and students without formaldehyde exposure (n = 51; P = 0.37). In conclusion, exposure of the medical students in western Europe to formaldehyde did not decrease MGMT activity in mononuclear blood cells.
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PMID:Activity of O6-methylguanine DNA methyltransferase in mononuclear blood cells of formaldehyde-exposed medical students. 1020 10


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