Cloning and expression of the Bacillus subtilis methyltransferase gene in Escherichia coli ada- cells

DNA fragments of Bacillus subtilis were inserted into a plasmid vector that can multiply in Escherichia coli cells, and foreign genes were expressed under the control of the lac promoter. By selecting hybrid plasmids that confer an increased resistance to alkylating agents on E. coli ada- mutant cells, the B. subtilis gene dat, which encodes O6-methylguanine-DNA methyltransferase, was cloned. The Dat protein, with a molecular weight of about 20,000, could transfer the methyl group from methylated DNA to its own protein molecule. Based on the nucleotide sequence of the gene, it was deduced that the protein comprises 165 amino acids and that the molecular weight is 18,779. The presumptive amino acid sequence of Dat protein is homologous to the sequences of the E. coli Ogt protein and the C-terminal half of the Ada protein, both of which carry O6-methylguanine-DNA methyltransferase activity. The pentaamino acid sequence Pro-Cys-His-Arg-Val, the cysteine residue of which is the methyl acceptor site in Ada protein, was conserved in the 3 methyltransferase proteins. The structural similarity of these methyltransferases suggests possible evolution from a single ancestral gene.     

Structural and immunological comparison of indigenous human O6-methylguanine-DNA methyltransferase with that encoded by a cloned cDNA

O6-Methylguanine-DNA methyltransferase, a ubiquitous and unusual DNA repair protein, eliminates mutagenic and cytotoxic O6-alkylguanine from DNA by transferring the alkyl group to one of its cysteine residues in a second-order suicide reaction. This 22-kDa protein was immunoaffinity-purified to homogeneity from cultured human lymphoblasts (CEM-CCRF line) and compared with the O6-methylguanine-DNA methyltransferase purified to homogeneity from Escherichia coli expressing a cloned human cDNA. The cellular and recombinant proteins were identical in size, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of intact molecules and their peptides. Immunoprobing of Western blots with three monoclonal antibodies specific for human cellular O6-methylguanine-DNA methyltransferase further indicated identity of the two proteins. The amino acid sequence of the cellular protein was experimentally determined for 87 out of a total of 207 residues and was found to be identical to that deduced from the cDNA sequence. A unique cysteine residue at position 145 was identified as the methyl acceptor site by autoradiographic analysis of peptides and sequence analysis of 3H-methylated O6-methylguanine-DNA methyltransferase. These observations establish that the cloned O6-methylguanine-DNA methyltransferase cDNA encodes the full-length O6-methylguanine-DNA methyltransferase polypeptide that is normally present in human cells. Moreover, the cellular protein does not appear to be significantly modified by posttranslational processes.     

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.     

O6-methylguanine-DNA methyltransferase levels in tissues of methionine-cysteine deficient subadult and adult mice

1. Subadult and adult mice were fed during 6 days a diet containing a complete mixture of amino acids or a mixture low in methionine-cysteine. 2. O6-methylguanine-DNA methyltransferase (MT) is the acceptor protein for methyl groups present in DNA at the O-6 position of a guanine that has been methylated by alkylating carcinogenic agents. 3. Upon methionine-cysteine deficiency O6-methylguanine-DNA methyltransferase levels decreased significantly in liver, but seemed unaffected in kidney, lung, testis and brain. 4. Age associated differences were found in liver, with lower values in the subadult than the adult mice leaving the young animals more vulnerable to exposure to alkylating agents. 5. To ensure an efficient repair of DNA lesions both age groups depended on a complete amino acid mixture in the diet.     

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.     

Site directed mutagenesis of two cysteine residues in the E. coli ogt O6-alkylguanine DNA alkyltransferase protein.

The E. coli ogt O6-alkylguanine-DNA alkyltransferase has two cysteine residues positioned identically with respect to cysteines in the E. coli ada O6-alkylguanine-DNA alkyltransferase. In order to assess their function, these residues were each substituted by a glycine to generate altered forms of the ogt protein. Mutagenesis of cysteine-139, located within a 'PCHRV' region of homology, eliminated functional activity confirming that this residue is the methyl-accepting cysteine in the active site of the protein. Substitution of cysteine 102 within the sequence 'LRTIPCG' had little effect on the ogt protein activity demonstrating that this cysteine is not directly involved with the transfer of O6-methylguanine adducts.     

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.     

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.     

中国人肿瘤细胞中O~6-甲基鸟嘌呤DNA甲基转移酶活性与对嘧啶亚硝脲敏感性的关系

 分析了20株中国人肿瘤细胞的O~6-甲基鸟嘌呤DNA甲基转移酶(O~6-MT)活性及对嘧啶亚硝脲ACNU的敏感性,发现两者间有较好的线性关系,O~6-MT低,对ACNU敏感,提示O~6-MT可作为使用ACNU对肿瘤化疗时的一项予见性指标。     

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.     

Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening

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.     

Repair of alkylated DNA in Escherichia coli. Physical properties of O6-methylguanine-DNA methyltransferase

An inducible methyltransferase of Escherichia coli acts on O6-methylguanine in DNA by conveying the methyl group to one of its own cysteine residues. The protein has now been purified to apparent homogeneity from a constitutively expressing strain. The homogeneous methyltransferase exhibits no DNA glycosylase or endonuclease activity on alkylated DNA. Further, the methyltransferase activity is strikingly resistant to heat inactivation under reducing conditions. The protein has Mr = 18,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient and Stokes radius of the native enzyme yield Mr = 18,400. The amino acid composition of the purified protein shows 4 to 5 cysteine residues/transferase molecule. The methylated, inactive form of the transferase has an unaltered molecular weight.     

Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening

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.     

Induction of SOS response,cellular efflux and oxidative stress response genes by chlorambucil in DNA repair-deficient Escherichia coli cells (ada,ogt and mutS)

Chlorambucil (CLB) is a bifunctional alkylating drug widely used as an anticancer agent and as an immunosuppressant. It is known to be mutagenic, teratogenic and carcinogenic. The cellular actions of CLB have remained poorly investigated. It is very likely that DNA damage and its repair are the key elements determining the destiny of CLB-exposed cells. We investigated the role of two specific DNA repair pathways involved in CLB-induced mutagenicity and gene expression changes by using Escherichia coli strains lacking either (i) two DNA methyltransferase functions (O(6)-methylguanine-DNA methyltransferase I (ada) and II (ogt)), or (ii) mismatch repair (MutS (mutS)). Mutagenicity was determined as the development of ciproxin and rifampicin resistance and the gene expression changes were assessed using expression profiling of all E. coli 4290 open reading frames (ORFs) by cDNA array. Chlorambucil-induced mutants in mutS cells, implying the importance of mismatch repair in preventing CLB-induced mutations. It also induced mutants in the ada, ogt strain, but to a lesser extent than in the wild-type strain. The simultaneous upregulation of several genes of the SOS response, cellular efflux and oxidative stress response, was demonstrated in both of the DNA repair-deficient strains but not in the wild-type cells. These and our previous results show that single-gene knock-out cells use specific gene regulation strategies to avoid mutations and cell death induced by agents such as chlorambucil.     

Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase.

Escherichia coli expresses two DNA repair methyltransferases (MTases) that repair the mutagenic O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT) DNA lesions; one is the product of the inducible ada gene, and here we confirm that the other is the product of the constitutive ogt gene. We have generated various ogt disruption mutants. Double mutants (ada ogt) do not express any O6MeG/O4MeT DNA MTases, indicating that Ada and Ogt are probably the only two O6MeG/O4MeT DNA MTases in E. coli. ogt mutants were more sensitive to alkylation-induced mutation, and mutants arose linearly with dose, unlike ogt+ cells, which had a threshold dose below which no mutants accumulated; this ogt(+)-dependent threshold was seen in both ada+ and ada strains. ogt mutants were also more sensitive to alkylation-induced killing (in an ada background), and overexpression of the Ogt MTase from a plasmid provided ada, but not ada+, cells with increased resistance to killing by alkylating agents. The induction of the adaptive response was normal in ogt mutants. We infer from these results that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the Ogt MTase also protects cells from killing by alkylating agents. We also found that ada ogt E. coli had a higher rate of spontaneous mutation than wild-type, ada, and ogt cells and that this increased mutation occurred in nondividing cells. We infer that there is an endogenous source of O6MeG or O4MeT DNA damage in E. coli that is prevalent in nondividing cells.     

Roles of two types of O6-methylguanine-DNA methyltransferases in DNA repair

Escherichia coli possesses 2 types of O6-methylguanine-DNA methyltransferases, one inducible and the other constitutive. These enzymes are coded by the ada and the ogt genes, respectively. Using a synthetic ogt-specific probe, we mapped ogt at 29.4 min, near the 5'-flanking region of the nirR gene, on the E. coli chromosome. To elucidate the roles of the 2 types of methyltransferases in DNA repair, we constructed mutant strains which lack either one or both of the genes. In either the ada+ or the ada- background, the ogt mutation had no effect on cell survival after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. On the other hand, ada- ogt- cells were more prone to mutation as compared to the ada- ogt+ cells exposed to MNNG. The frequency of spontaneous mutation of cells defective in either one or both of the genes was the same, however, the introduction of the ogt+ plasmid into the cells produced a 2-3-fold decrease in the frequency of spontaneous mutation. O6-Methylguanine-DNA methyltransferases appear to eliminate premutagenic DNA lesions not only from cells exposed to alkylating agents but also from those grown in the absence of the agents.