Treatment of Escherichia coli cells with alkylating agents results in the induction of a set of genes that repair alkylated DNA. The genes known to be induced are ada, alkA, alkB, and aidB. They share a common regulatory pathway and are collectively called the adaptive response genes. Induction occurs when Ada protein becomes activated by methylation as a consequence of its repair activity. Activated Ada protein then functions as a positive regulatory element that stimulates transcription of the adaptive response genes. The aidB gene is the least well understood component of the adaptive response. This proposal outlines our efforts to examine three major features of the E.coli aidB gene. 1) We will examine its Ada- dependent induction upon alkylation treatment by characterizing its promoter-operator DNA sequences and examining the interaction of the Ada protein with its regulatory binding site. The mechanism by which Ada mediates aidB induction will be compared with the molecular mechanisms of ada and alkA regulation. 2) In addition to its regulation by Ada, the aidB gene is subject to a second form of regulation, aidB is induced when cells are grown anaerobically. Anaerobic induction is unique to aidB is not shared by other adaptive response genes and appears to function by a regulatory mechanism that is independent of Ada. We will examine this second regulatory pathway and determine the molecular mechanisms by which it functions. This aspect of the study will be approached in two ways. We will determine the aidB regulatory DNA sequences required for its anaerobic induction and identify the base pairs within the regulatory region required for this type of induction. This will be done by fusion of a fragment of aidB known to contain both anaerobic and alkylation dependent upstream regulatory sequences to a lacZ reporter gene residing on a plasmid. We will first localize the regulatory regions by deletion analysis, identify the critical components within this region by mutagenesis. After mutagenesis we will select mutants that exhibit altered aidB expression, then identify the altered base pairs by DNA sequence analysis. The second approach to understanding the anaerobic regulatory pathway will be to isolate and characterize trans-acting mutants that are altered in their ability to express aidB during anaerobiosis. This will allow us to determine the genes involved in the anaerobic regulation and to characterize the genetic pathway leading to induction. 3) We will characterize the aidB gene, or operon, by identifying its products and by cloning and sequencing the gene. We will also examine the role of these gene products in alkylation resistance both in aerobically- and anaerobically-growing cells by examining the effects of mutations in this gene on repair of alkylated DNA and/or production of lesions by alkylating agents whose metabolic activation may require aidB function. This aspect of the project will also allow us to examine the high degree of resistance to alkylation treatments afforded by anaerobiosis and possible role of aidB in modulating this high level resistance.
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