Mechanisms of mutagenesis in " hot spots" causing cancer

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DESCRIPTION (provided by applicant): The long-term goal of these investigations is to use two microbial model systems to determine the mechanisms by which gene transcription and supercoiling triggered by stress increase mutation rates specifically in the activated genes. These mutations occur in the same kinds of secondary structures known to cause cancer. Recent evidence from our laboratory suggests that, in both microbes and man, transcription drives DNA supercoiling which creates stem-loop structures (SLS) with unpaired bases vulnerable to mutations. Stressors can also affect negative supercoiling directly, resulting in more SLS and higher mutation rates. In our E. coli model system we will determine the effects of stress on transcription, supercoiling, and mutation frequency, obtain evidence for the existence of predicted SLS in both transcribed and non-transcribed strands, and modify sequences in SLS to examine predicted effects on mutation rates. In our S. cerevisiae model system, we will examine the effects of various kinds of environmental stressors on reversion rates of specific hypermutable bases of the p53 cancer gene. Using a DNA folding program and a new computer algorithm we have localized cancer-causing bases in DNA SLS and successfully predicted their mutability indexes (MIs). With the yeast-plasmid model we hope to verify our method for predicting mutation frequencies and advance our understanding of the variables that cause mutations. For example, we will compare known structures, MIs and forward (wild to mutant) mutation frequencies inp53 hypermutable and control bases to those of the backward (mutant to wild) mutations, in the same SLS. We will compare the effects of transcription, supercoiling, and stressors on mutation frequencies of bases that differ in MIs and locations in SLS: The effects of known carcinogens on reversion rates of different bases in the same vulnerable position of a SLS can be compared, to clarify the relative importance of 1) the base per se, vs 2) the location of the base in determining mutation frequencies. These investigations should provide new insights into the mechanisms of mutagenesis and carcinogenesis.