Inactive O6-methylguanine-DNA methyltransferase in human cells.

A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.     

Physicochemical studies of human O6-methylguanine-DNA methyltransferase

O6-Methylguanine-DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O6-alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second-order rate constants of 2.20 x 10(8) and 0.067 x 10(8) lmol-1 min-1 at 37 degrees C for duplex and single-stranded DNA substrates, respectively. The corresponding value for the alkylated base in synthetic poly(dC, dG, m6dG) is 0.02 x 10(8) l mol-1 min-1. The native protein is monomeric with a molecular mass of 22-24 kDa. Methylation of the protein does not lead to a gross change in its conformation but causes a slight reduction in its isoelectric point of 6.2. Although DNA protects the protein from heat inactivation, both duplex and single-stranded DNAs inhibit its activity in a concentration-dependent manner. The transferase reaction rate is also strongly inhibited by salt with about 20% of the maximum rate observed in physiological ionic strength. This inhibition is nonspecific with respect to the ions of univalent salts.     

O6-methylguanine-DNA methyltransferase content in human lymphocytes following surgical trauma

O6-methylguanine-DNA methyltransferase removes methyl groups from the O-6 position of guanine in DNA previously alkylated by alkylating carcinogens. Thus, the protein facilitates restoration of the impaired DNA. The content of O6-methylguanine-DNA methyltransferase was assayed in circulating lymphocytes and the impact of surgical trauma investigated. Patients (n = 13) without metabolic diseases admitted for elective orthopedic surgery were used. The patients were allowed water and food postoperatively. Blood was taken before and 3 days following surgery and the circulating lymphocytes were isolated. Before surgery, the O6-methylguanine-DNA methyltransferase content determined in the cell extracts showed patient-specific variations. Following surgery, a significant decrease of the protein by 60% (from 609 to 243 fmole/mg of DNA) was observed. The intensity of surgical trauma was confirmed by the decrease in plasma albumin concentration and the increase in white blood cell counts. The surgical trauma might elicit its effect as either a change in turnover of O6-methylguanine-DNA methyltransferase or a release from the thymus of lymphocytes low in enzyme levels. In summary, the surgical trauma per se was the cause of the pronounced decrease in the O6-methylguanine-DNA methyltransferase seen here. Investigations on O6-methylguanine-DNA methyltransferase levels have an important relevance in studies on tumor-promoting agents inhaled and then taken up by the T lymphocytes of prospective proliferating capacity.     

Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase

The O6-methylguanine-DNA methyltransferase of Escherichia coli acts rapidly and stoichiometrically to convert a mutagenic O6-methylguanine residue in DNA to unsubstituted guanine. Even at low protein concentrations and in the absence of any cofactors, the transfer of a methyl group to one of the protein's own cysteine residues occurs in less than 2 s at 37 degrees C. The entire kinetic process can be followed experimentally at 5 degrees C. Formation of S-methylcysteine in the protein is accompanied by loss of activity and accounts for the exceptional suicide kinetics of this enzyme as well as for the sharp saturation of O6-methylguanine repair observed in vivo. The enzyme can remove greater than 98% of the methyl groups from O6-methylguanine present in alkylated DNA, but leaves N-alkylated purines untouched. Single-stranded DNA containing O6-methylguanine is a poor substrate, with the methyl transfer occurring at approximately 0.1% of the rate for duplex DNA. This latter observation may explain the high frequency of mutations induced by alkylating agents at DNA replication forks.     

Quantitation of O6-methylguanine-DNA methyltransferase in HeLa cells

A synthetic DNA polymer containing [8-³H]O⁶-methylguanine m⁶G) was used as a substrate to assay the in situ demethylation of the alkylated base by an activity in HeLa cell extracts. The repair activity appears to be similar to the O⁶-methylguanine-DNA methyltransferase of E. coli and to be inactivated by reaction with the substrate. Extracts of a methylation-repair proficient (Mer⁺) cell strain, HeLa CCL2, were found to contain m⁶G repair activity equivalent to approx. 100 000 molecules of methyltransferase per cell, assuming that each molecule can demethylate one m⁶G residue. No activity could be detected in the extract of a repair deficient (Mer⁻) cell strain, HeLa S3, and there is no evidence of an inhibitor of repair activity in this strain.     

A constitutive O6-methylguanine-DNA methyltransferase of Rhizobium meliloti

We have identified a DNA methyltransferase activity of the nitrogen-fixing bacterium, Rhizobium meliloti, that repairs O6-methylguanine lesions. Repair of the O6-methylguanine residue results in transfer of the methyl group to a cysteine residue of a 28,000-dalton protein. The O6-methyltransferase activity is expressed constitutively and R. meliloti does not exhibit an adaptive response to alkylating agents.     

Crystallization of O6-methylguanine-DNA methyltransferase from Escherichia coli

The 19,000 Mr C-terminal domain of the Escherichia coli ada gene product that contains O6-methylguanine-DNA methyltransferase DNA repair activity has been crystallized in a low-salt environment. The crystals, which diffract to 2.3 A (1 A = 0.1 nm), are suitable for detailed structural studies. The space group is P21 with unit cell dimensions a = 46.3 A, b = 45.8 A, c = 46.9 A and beta = 113.3 degrees.     

Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.     

Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase

The level of O6-methylguanine-DNA methyltransferase activity in a human cell line carrying a 1.1-kilobase cDNA fragment was about 50 times higher than that found in ordinary methyltransferase-proficient (Mer+) cell lines (Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Biol. 213, 739-747). Taking advantage of this overproduction, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. A single polypeptide with a molecular weight of approximately 25,000 was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most highly purified preparation. The Stokes radius of 22.5 A and the sedimentation coefficient of 2.0 S were obtained, from which the molecular weight of the native form of the enzyme was calculated to be 19,000. After digestion with lysyl endopeptidase, peptide fragments of the protein were isolated and sequenced. The amino acid sequences of these peptides and the amino acid composition of the protein were in good agreement with those deduced from the nucleotide sequence of the cloned cDNA. The purified enzyme catalyzed transfer of methyl groups from O6-methylguanine and O4-methylthymine, but not from methylphosphotriesters, of methylated DNA to the enzyme molecule.     

Cloning and expresion of cDNA for rat O6-methylguanine-DNA methyltransferase.

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.     

Active site amino acid sequence of the bovine O6-methylguanine-DNA methyltransferase.

An O6-methylguanine-DNA methyltransferase has been partially purified from calf thymus by conventional biochemical techniques. The enzyme was specifically radioactively labelled at the cysteine residue of the active site and further purified by attachment to a solid support. Following digestion with trypsin, a radioactive peptide containing the active site region of the protein was purified by size fractionation, ion exchange chromatography and reverse phase HPLC. The technique yielded an essentially homogeneous oligopeptide which was subjected to amino acid sequencing. The sequence adjacent to the acceptor cysteine residue of the bovine protein exhibits striking homology to the C-terminal methyl acceptor site of the E. coli Ada protein and the proposed acceptor sites of the E. coli Ogt and the B. subtilis Dat1 proteins.     

Characterization of human cytoglobin gene promoter region

Cytoglobin (CYGB) is a member of the vertebrate globin family together with hemoglobin, myoglobin and neuroglobin. Although the physiological function of CYGB is still unclear, spectroscopic studies show that CYGB contains a hexacoordinated heme pocket similar to other pentacoordinated globin proteins. CYGB shares a common phylogenetic ancestry with vertebrate myoglobin from which it diverged by duplication before the appearance of jawed vertebrates. The objective of this study is to identify the regulatory and promoter region of the human cytoglobin gene. 5' unidirectional deletion constructs demonstrated that the proximal promoter elements of human CYGB gene are located between -1113 to -10 relative to the translation start site. Site-directed mutagenesis showed that mutation of a c-Ets-1 motif at -1008 and Sp1 motifs at -400, -230 and -210 remarkably decreased the promoter activity. Gel shift assays confirmed the binding of DNA-nuclear proteins to these motifs. All these results indicate that CYGB gene expression can be up-regulated by c-Ets-1 and Sp1 motifs.     

Different patterns of DNA methylation of the two distinct O6-methylguanine-DNA methyltransferase (O6-MGMT) promoter regions in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal cancer incidence differs widely among different geographic regions. In addition to mutational changes, epigenetic mechanisms also play important roles in the pathogenesis of CRCs. O6-methylguanine-DNA methyltransferase (O ⁶ -MGMT) is a DNA repair protein and in the absence of MGMT activity, G-to-A transition may accumulate in the specific genes such as K-ras and p53. To identify which CpG sites are critical for its downregulation, we analyzed the methylation status of the MGMT gene promoter in two sites in CRC patients. Then we compared the frequency of their methylation changes with the results of our previously reported K-ras gene mutation, APC2 and p16 methylation. MGMT methylation was examined in 92 tumor samples. A methylation specific PCR (MSP) method was performed for two loci of MGMT gene which described as MGMT-A and MGMT-B. The prevalence of MGMT-A, and MGMT-B methylation was 49/91 (53.8 %), and 83/92 (90.2 %), respectively. We detected high frequency of MGMT-B but not MGMT-A methylation in tumor tissues with APC2 methylation. Our results showed that MGMT-B methylation is significantly associated with K-ras gene mutation rather than MGMT-A (p = 0.04). Simultaneously, an inverse correlation was found between p16 and MGMT-B methylation simultaneously (p = 0.02). Our study indicated that hypermethylation of the specific locus near the MGMT start codon is critical for cancer progression. MGMT-B assessment that is associated with K-ras mutation can have a prognostic value in patients with CRC.     

Polyclonal antibodies against O6-methylguanine-DNA methyltransferase in adapted bacteria

The similarity of the adaptive response and the methyltransferase component in bacterial strains from different phylogenic groups was investigated. An adaptive response with induction of transferase activity was found for the first time in the soil bacteria P. aeruginosa and X. maltophilia. Polyclonal antibodies against the E. coli ada protein were used to investigate the structural similarity of the transferases from several bacterial strains with adaptive responses and inducible transferase activity. These antibodies cross-reacted with transferase from M. luteus and P. aeruginosa but not with proteins from other related bacteria, and not with human transferase. The phylogenic relationships of bacteria with adaptive responses suggest that the response likely was present in the common ancestor of eubacteria. The restricted antibody cross-reactivity may reflect the dual role of the E. coli ada protein not only in DNA repair but in positive gene regulation.     

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli.

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.     

Proteomic analysis of human O6-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

Recent evidence suggests that human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase delta, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21(waf1/cip1)), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1alpha), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90alpha and beta, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.