The goal of this project is to further understanding of how short-lived reactive oxidizing species (ROS) generated outside the nucleus nevertheless lead to the induction of gene locus mutations. To accomplish this, this project will investigate quantitative and qualitative aspects of the induction of gene mutations by ROS generated outside the nucleus, and compare them to mutations generated by ROS within the nucleus. ROS will be produced uniformly within the cell or targeted to non-nuclear regions by using photo-activated dyes that concentrate in specific sub-cellular compartments. Quantitative and qualitative measurements of DNA damage will be made using the comet, or single ceil gel electrophoresis assay. The mutagenicity studies will involve a two-pronged approach. First, quantitative dose-response experiments will be carried out at two endogenous gene loci, and measured as a function of damage to DNA. Second, the qualitative natures of induced mutants will be determined by analyses of mutational spectra induced by the various treatments. These studies will be done initially in human lymphoblastoid cells with a mutated p53 gene. An additional set of experiments will address the question of whether mutagenic effects of ROS exposure are dependent on p53 status. The same strategies described above will be repeated using p53 wild type and null cells from the same donor. Finally, another set of experiments will examine effects of the Fanconi's anemia complex on ROS mutagenesis.
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