Zinc binding by the methylation signaling domain of the Escherichia coli Ada protein.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.     

Mutant Escherichia coli Ada proteins simultaneously defective in the repair of O6-methylguanine and in gene activation.

The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also possesses two distinct suicide alkyltransferase activities, for O6-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair O6-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal O6-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.     

Characterization of the major DNA repair methyltransferase activity in unadapted Escherichia coli and identification of a similar activity in Salmonella typhimurium.

Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.     

The kinetics of the alkaline hydrolysis of phosphotriesters in DNA.

The degradation in alkali of normal DNA and DNA alkylated with dimethyl sulphate (DMS), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) has been investigated using analytical ultracentrifugation techniques. For control T7-DNA (w.st. denatured form 12.5 - 10(6) daltons) the rate of degradation at 37 degrees varies from 0.14 breaks/molecule/h in 0.1 M NaOH to 1.2 breaks/molecule/h in 0.4 M NaOH. When DNA is alkylated with reagents known to produce phosphotriesters addition of alkali leads to an initial rapid degradation not observed with control DNA. Ethyl phosphotriesters are hydrolysed at about half the rate of methyl phosphotriesters. Approximately one third of the methyl or ethyl phosphotriesters present hydrolyse to give breaks in the DNA chain.     

Methyl transferases induced during chemical adaptation of M. luteus.

Three peaks of methyltransferase activity specific for MNNG alkylated DNA have been identified from extracts of chemically adapted M. luteus. They are designated as TI to TIII in order to their elution from a Sephadex G-75 column. The first one of these peaks has been purified to homogeneity. TI, is an inducible, unusually salt resistant, heat labile protein which corrects O6-methylguanine in alkylated DNA by the transfer of the O6-alkyl group to a cysteine amino acid in the TI protein. There is a stoichiometric relationship between the loss of O6-methylguanine from the DNA and the production of S-methylcysteine. Partially purified TII & TIII proteins show specificity for O4-alkylthymine and methyl phosphotriesters respectively. The mode of repair by the isolated methyltransferases is similar yet there is no competition for substrate specificity. The apparent molecular weights of TI, TII & TIII proteins are 31Kd, 22Kd, and 13Kd respectively.     

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.     

Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included.     

An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo.

Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.     

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.