The goal of this research project is to gather new information about the process of mutation induction in Escherichia coli and Salmonella typhimurium. The recent observations that simple base pair substitutions may be sufficient for activating normal cellular genes of unknown function to oncogenes, and the well established correlation between mutagenicity and carcinogenicity, suggests that attempts to understand the molecular basis for mutation induction in bacteria may be relevant to our comprehending at least one of the steps leading to the conversion of normal cells to cancer cells. The project will focus on the mutagenic and lethal effects of treating bacteria wih isopsoralen plus near-UV and will use genetic loci cloned into the single stranded phages M13 and f1 to find answers to the following questions: 1. does the SOS-repair activity encoded by umuDC operate equally well on single stranded and double stranded DNA? 2. are mutations induced by isopsoralen plus near-UV treatment arising from damage to pyrimidine bases, purine bases, or both? 3. is the DNA tetranucleotide sequence 5'-TATA-3' a hotspot for mutagenesis by isopsoralen plus near-UV treatment? 4. what is the fidelity of nick-translation of DNA that has been damaged by isopsoralen plus near-UV treatment? 5. what is the base mismatch specificity of methyl-instructed mismatch repair? In addition to addressing these specific questions, a broad goal of the project is to isolate and characterize new alleles of the mucAB locus of the plasmid pKM101. Attempts will be made to identify temperature sensitive and nonsense mutations in muc which could be exploited to investigate the biochemical basis for repair and mutagenesis functions encoded by this locus.
