Hughes, Stephen J.; (1990) Molecular Studies on the Regulation of the Adaptive Response to Alkylating Agents in Escherichia coli. Doctoral thesis (Ph.D), UCL (University College London).

Text Molecular studies on the regulation of the adaptive response to alkylating agents in Escherichia coli.pdf Download (6MB)

Abstract

When Escherichia coli cells are exposed to low, non-lethal doses of simple alkylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N- nitrosourea, they acquire an increased resistance to the mutagenic and cytotoxic effects of a subsequent challenge with a high dose of these agents. This cellular response, referred to as the adaptive response to alkylating agents, results from the increased expression of at least four genes, ada, alkA, alkB and aidB. The products of the ada and alkA genes are DNA repair enzymes that remove potentially mutagenic and cytotoxic alkylation lesions from cellular DNA. The Ada protein also acts as a positive regulator of expression of the inducible genes of the adaptive response. In this study the regulatory ada gene has been cloned from four independently- isolated E. coli mutants which express the adaptive response constitutively in the absence of alkylation damage. Nucleotide sequence analysis has shown that each gene contains two G-C to A-T transition mutations within the coding region which result in the synthesis of a mutant Ada protein containing two amino acid substitutions in the N-terminal region. Three of the mutant strains have the same two ada mutations. E. coli transformed with recombinant plasmids carrying the mutated ada genes overexpressed both the mutated ada gene and the chromosomal alkA gene, indicating that the mutant Ada proteins are strong inducers of ada and alkA gene expression in vivo. The Ada protein purified from one of the constitutive mutants was also shown to be a strong inducer of expression of the wild-type ada gene in an in vitro coupled transcription-translation system. One amino acid substitution, methionine-126 substituted by isoleucine, occurred in the Ada protein synthesized by all four mutant strains, and this substitution alone has been shown to be sufficient to convert the Ada protein into a strong activator of ada and alkA gene expression in vivo. Models for the mechanism by which this substitution exerts its effect on gene expression are presented.

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