The adaptive response to alkylation damage. Constitutive expression through a mutation in the coding region of the ada gene

We have shown by genetic mapping, molecular cloning, and DNA sequencing that four Escherichia coli mutants, which express the adaptive response to alkylation damage constitutively, are mutated in the ada gene. All four mutant ada genes have two GC to AT transition mutations in the coding region and encode altered Ada proteins with two amino acid substitutions in the N-terminal domain. E. coli carrying the mutated ada genes on recombinant plasmids overexpressed both the mutated ada gene and the chromosomal alkA gene. This observation indicates that the mutant Ada proteins act as strong positive regulators of the ada and alkA genes in the absence of DNA alkylation. One mutant protein, Ada-11, was shown to be a strong activator of ada gene expression in a cell-free system. An altered pattern of tryptic digestion of the Ada-11 protein compared with the wild-type Ada protein suggested that it has a different conformation. One amino acid substitution, namely methionine residue 126 replaced by isoleucine, occurred in all four mutant Ada proteins, and this mutation alone was sufficient to convert the Ada protein into a strong activator of ada and alkA gene expression in vivo.     

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.