Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA lesions that halt RNA polymerase during transcription are preferentially repaired by the nucleotide excision repair pathway. This transcription-coupled repair is initiated by the arrested RNA polymerase at the DNA lesion. However, the mutagenic O6-methylguanine (6MG) lesion which is bypassed by RNA polymerase is also preferentially repaired at the transcriptionally active DNA. We report here a plausible explanation for this observation: the human 6MG repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is present as speckles concentrated at active transcription sites (as revealed by polyclonal antibodies specific for its N and C termini). Upon treatment of cells with low dosages of N-methylnitrosourea, which produces 6MG lesions in the DNA, these speckles rapidly disappear, accompanied by the formation of active-site methylated MGMT (the repair product of 6MG by MGMT). The ability of MGMT to target itself to active transcription sites, thus providing an effective means of repairing 6MG lesions, possibly at transcriptionally active DNA, indicates its crucial role in human cancer and chemotherapy by alkylating agents.
Mol Cell Biol 1998 Mar
PMID:Implication of localization of human DNA repair enzyme O6-methylguanine-DNA methyltransferase at active transcription sites in transcription-repair coupling of the mutagenic O6-methylguanine lesion. 948 83

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.
Mol Gen Genet 1998 Apr
PMID:The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme. 961 74

O6-methylguanine-DNA methyltransferase (MGMT) is a major determinant of susceptibility to methylating carcinogens and of tumor resistance to anticancer methylating and chloroethylating drugs. The silencing of MGMT expression that occurs in 20-30% of human tumor lines is tightly linked to methylation within the MGMTgene 5'CpG island. Previous studies on a very limited number of cell lines showed that such methylation was uneven, with hot-spots where methylation almost invariably occurred and intervening regions with very low incidences of methylation. To ascertain if such hot-spot methylation is in fact a ubiquitous hallmark of MGMT-silenced cells, we determined the methylation status of selected hot-spot CpGs in an extensive panel of MGMT-expressing and -silenced cell lines and xenografts. Using two simple and rapid bisulfite-polymerase chain reaction-based assays, we confirmed that in MGMT-silenced cells, methylation occurred at these sites whereas it was essentially absent in MGMT-expressing cells.
Mol Carcinog 1999 Feb
PMID:Methylation of selected CpGs in the human O6-methylguanine-DNA methyltransferase promoter region as a marker of gene silencing. 1007 35

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation-sensitive restriction enzymes (BssHII, SacII, Eagl, Nael, and Smal) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of Eagl and Nael sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P<0.01). Methylation-sensitive single-nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by Eagl and Nael digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the Eagl and Nael sites and nt -128 than in the 13 tumors lacking the methylation pattern (P<0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression.
Mol Carcinog 1999 Feb
PMID:A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers. 1007 36

The crystal structure of O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) of hyperthermophilic archaeon Pyrococcuskodakaraensis strain KOD1 (Pk -MGMT) was determined by single isomorphous replacement method with anomalous scattering (SIRAS) at 1.8 A resolution. The archaeal protein is extremely thermostable and repairs alkylated DNA by suicidal alkyl transfer from guanine O6 to its own cysteine residue. Archaea constitute the third primary kingdom of living organisms, sharing characteristics with procaryotic and eucaryotic cells. They live in various extreme environments and are thought to include the most ancient organisms on the earth. Structural studies on hyperthermophilic archaeal proteins reveal the structural features essential for stability under the extreme environments in which these organisms live, and will provide the structural basis required for stabilizing various mesophilic proteins for industrial applications. Here, we report the crystal structure of Pk-MGMT and structural comparison of Pk-MGMT and methyltransferase homologue from Escherichia coli (AdaC, C-terminal fragment of Ada protein). Analyses of solvent-accessible surface area (SASA) reveals a large discrepancy between Pk-MGMT and AdaC with respect to the property of the ASA. In the Pk-MGMT structure, the intra-helix ion-pairs contribute to reinforce stability of alpha-helices. The inter-helix ion-pairs exist in the interior of Pk-MGMT and stabilize internal packing of tertiary structure. Furthermore, structural features of helix cappings, intra and inter-helix ion-pairs are found around the active-site structure in Pk-MGMT.
J Mol Biol 1999 Sep 24
PMID:Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal O6-methylguanine-DNA methyltransferase. 1049 33

Gene therapy has been proposed to have implications in the treatment of cancer. By genetically manipulating the hematopoietic stem cell compartment with genes that confer resistance to chemotherapeutic agents, the dose escalation that is necessary to effectively treat the cancers could potentially be achieved. DNA repair genes are some of the potential candidates to confer increased resistance to chemotherapeutic agents. Although initial focus in this area has been on the direct reversal protein (MGMT), its protective ability is limited to those agents that produce O(6)-methylGuanine cross-links-agents that are not extensively used clinically (e.g., nitrosoureas). Furthermore, most alkylating agents attack more sites in DNA other than O(6)-methylGuanine, such that the protections afforded by MGMT may prevent the initial cytotoxicity, but at a price of increased mutational burden and potential secondary leukemias. Therefore, some of the genes that are being tested as candidates for gene transfer are base excision repair (BER) genes. We and others have found that overexpression of selective BER genes confers resistance to chemotherapeutic agents such as thiotepa, ionizing radiation, bleomycin, and other agents. As these "proof of concept" analyses mature, many more clinically relevant chemotherapeutic agents can be tested for BER protective ability.
Environ Mol Mutagen 2000
PMID:DNA repair and gene therapy: implications for translational uses. 1071 40

A significant fraction of human cancers are thought to have a genetic component and several lines of evidence suggest that deficiencies in DNA repair may be a contributing factor. Little is known, however, about the frequency and distribution of variants of DNA repair genes in the general human population. The protein truncation test (PTT) was used to screen 136 healthy volunteers for protein-truncating variants of 10 DNA repair genes: APE, CDK7, ERCC1, WAF1, HOGG1, MGMT, POLB, UNG, HAAG, and CCNH. This sample consisted of 41males (30%) and 95 females (70%) with an average age of 25.3 years, ranging from 17 to 60 years of age. No truncating mutations were found in the 10 genes examined in any of the subjects. The 95% confidence interval for a proportion of 0 over the 272 alleles examined per locus is 0-0.01. The calculated frequency of truncating mutations in each of these genes, among the general population, is thus less than 1%. Among the 10 genes tested in 136 people, a single sample had no PCR product for HAAG, even though PCR products were obtained on all other genes. Total RNA dot hybridization confirmed the presence of HAAG mRNA transcripts in this sample. Despite identification of this single DNA repair variant, these results indicate a low frequency of truncating mutations in DNA repair genes in the general population.
Environ Mol Mutagen 2000
PMID:Screening a human population sample for DNA repair gene deficiencies utilizing the protein truncation test. 1104 4

Transfer of drug resistance genes to hematopoietic stem cells offers the potential to protect cancer patients from drug-induced myelosuppression and to increase the number of gene-modified cells by in vivo selection. In this study, a retroviral vector expressing both a P140K variant of human O6-methylguanine-DNA methyltransferase (MGMT) and an EGFP reporter gene was evaluated for stem cell protection in a murine transplant model. Mice transplanted with vector-transduced cells showed significant resistance to the myelosuppressive effects of temozolomide (TMZ), an orally administered DNA-methylating drug, and O6-benzylguanine (BG), a drug that depletes cells of wild-type MGMT activity. Following drug treatment, increases in EGFP(+) peripheral blood cells were seen in all peripheral blood lineages, and secondary transplant experiments proved that selection had occurred at the stem cell level. In a second set of experiments in which transduced cells were diluted with unmarked cells, efficient stem cell selection was noted together with progressive marrow protection with repeated treatment courses. Altogether, these results show that P140K MGMT gene transfer can protect stem cells against the toxic effects of TMZ and BG and that this vector/drug system may be useful for clinical myeloprotection and for in vivo selection of transduced stem cells.
Mol Ther 2001 Jan
PMID:Protection and in vivo selection of hematopoietic stem cells using temozolomide, O6-benzylguanine, and an alkyltransferase-expressing retroviral vector. 1116 14

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.
Am J Physiol Lung Cell Mol Physiol 2001 Apr
PMID:Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase. 1123 17

Cell proliferation requires precise control to prevent mutations from replication of (unrepaired) damaged DNA in cells exposed spontaneously to mutagens. Here we show that the modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (R-MGMT), formed from the suicidal repair of the mutagenic O(6)-alkylguanine (6RG) lesions by MGMT in the cells exposed to alkylating carcinogens, functions in such control by preventing the estrogen receptor (ER) from transcription activation that mediates cell proliferation. This function is in contrast to the phosphotriester repair domain of bacterial ADA protein, which acts merely as a transcription activator for its own synthesis upon repair of phosphotriester lesions. First, MGMT, which is constitutively present at active transcription sites, coprecipitates with the transcription integrator CREB-binding protein CBP/p300 but not R-MGMT. Second, R-MGMT, which adopts an altered conformation, utilizes its exposed VLWKLLKVV peptide domain (codons 98 to 106) to bind ER. This binding blocks ER from association with the LXXLL motif of its coactivator, steroid receptor coactivator-1, and thus represses ER effectively from carrying out transcription that regulates cell growth. Thus, through a change in conformation upon repair of the 6RG lesion, MGMT switches from a DNA repair factor to a transcription regulator (R-MGMT), enabling the cell to sense as well as respond to mutagens. These results have implications in chemotherapy and provide insights into the mechanisms for linking transcription suppression with transcription-coupled DNA repair.
Mol Cell Biol 2001 Oct
PMID:The modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase is a negative regulator of estrogen receptor-mediated transcription upon alkylation DNA damage. 1156 93


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