Molecular mechanisms of the adaptive response to alkylating agents

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Exposure of Escherichia coli cells to relatively low levels of alkylating agents leads to the formation of enzymes related to the repair of alkylation-associated DNA damage. Once these enzymes are produced, the cell becomes more resistant to both the killing and mutagenic effects of various alkylating agents. This process is termed the "adaptive response". In this process at least four genes, ada, alkA, alkB, and aidB, are known to be involved, among which the ada gene plays a central role. Ada protein, the product of the ada gene, acts as a transcriptional regulator for its own gene and for others belonging to this regulon. The Ada protein carries two distinct methyltransferase activities, one transfers a methyl group from one of two stereoisomers of methylphosphotriesters of alkylated DNA to a specific cysteine residue (Cys69) of the enzyme and the other transfers a methyl group from an O^6-methylguanine or O^4-methylhymine residue of the DNA to a cysteine residue (Cys321) located cl … More ose to the C-terminus of the protein.Expression of the alkA gene in Escherichia coli is controlled by Ada protein, which binds to a specific region of the alkA promoter and enhances further binding of RNA polymerase holoenzyme to the complex. To determine the sequence recognized by the Ada protein, we introduced various base substitutions into the promoter region of alkA and examined their effects on expression of the gene, both in vivo and in vitro. Base changes within the sequence AAAGCAAA, located between positions -41 and -34 from the transcription initiation site, greatly decreased the frequencies of initiation of transcription. In footprinting experiments, the region containing this sequence was protected by the Ada protein and base change within this sequence let to failure of binding of Ada protein to the promoter. It is likely that the Ada protein recognizes the AAAGCAAA sequence in the alkA promoter and binds to the region containing the sequence, thereby allowing ready access of RNA polymerase to the promoter. There are considerable differences between the mechanisms of action of Ada protein on the promoters of alkA and ada, even though the expression of both genes is positively relulatoed by Ada protein. Less