In the bacteriun E. coli, a complex and coordinate response to chemical mutagen treatment, termed the 'SOS' response, involves alterations of the transcription of more than 15 diverse genes. My laboratory has begun a molecular analysis of a DNA damage response (DDR) in the unicellular eucaryote Saccharomyces cerevisiae which may be a counterpart to the 'SOS' regulon. We have isolated a family of more than 10 genes which show altered transcription following exposure to a wide variety of mutagens/carcinogens. We propose investigating the molecular details of the regulation of two of these genes, DDRA2 and DDR48. In order to identify the sequences required for DNA damage (and heat shock) regulation, a series of deletions will be constructed using BAL31 exonuclease that remove increasing amounts of the 5' upstream regions. The effects of these deletions on transcript regulation will be assessed by Northern hybridization analysis. Gel retardation assays will be used to investigate binding of proteins to important upstream regions. The sites of protein-DNA contact will be determined by performing 'footprinting' on the gel retarded fragment. We will also examine changes in chromatin organization surrounding the DDR genes following DNA damage or heat shock using DNAaseI hypersensitivity experiments. Antibodies to the DDR gene products will be prepared in order to investigate the localization of the DDR proteins in damaged cells. Finally, we will investigate the stimulation of Ty transposition by DNA damage using both genetic and biochemical probes.
