The goal of the proposed research is to understand the mechanism and regulation of cellular responses to DNA damage. Exposure of Escherichia coli to a variety of carcinogens and mutagens elicits a diverse array of cellular responses often termed the SOS functions. A single step operon fusion technique will be used to fuse the beta-galactosidase gene to the regulatory regions of din (damage-inducible) genes. In these fusion strains beta-galactosidase has been shown to be inducible by ultraviolet light and mitomycin C in a recA plus lexA plus -dependent fashion. The regulation of these din genes will be studied by introducing other mutations which affect DNA repair and by isolating new mutations. The effect of a variety of treatments and conditions on the induction of beta-galactosidase will be examined in an effort to determine the cellular processes and signals controlling the expression of the SOS functions. The functions of the din genes will be deduced by examining the phenotypes of the fusion strains and by assaying for relevant enzymatic activities. Specialized transducing phage and small plasmids will be used to isolate the regulatory regions of the din genes. Molecular aspects of regulation will then be studied by sequencing these regions and examining their interaction with possible regulatory proteins. Specific results of the above studies will be used to improve the sophistication of short term bacterial genetic toxicity tests.
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