Our overall goal is to understand how eukaryotic cells protect against the toxic and mutagenic effects of DNA damaging agents, in particular alkylating agents. Such mutagens and carcinogens are everpresent in our environment as both synthetic and naturally occurring compounds. In addition, numerous alkylating agents are commonly used in the cancer clinic for chemotherapy. Given that one in three people will be diagnosed with cancer at some point during their lifetime (at least in the Western world) large numbers of people will be exposed to very toxic levels of these agents. A clear understanding of how normal cells and tumor cells respond to alkylating agents is therefore important. We have studied the cellular responses to alkylating agents in E. coli, S. cerevisiae, S. pombe, rodent and human cells. Indeed, using cloned alkylation repair genes we have genetically manipulated these organisms to understand the relative importance of responses to different types of alkylated DNA bases. During the last funding period we generated two new strains of mice that are null for the Aag 3MeA DNA glycosylase and the Mgmt DNA repair methyltransferase alkylation repair genes. We also characterized a novel mechanism by which mutator phenotypes can be generated, and we analyzed the global transcriptional response of S. cerevisiae to an alkylating agent using DNA chip technology. In addition we demonstrated that the 3MeA and O6MeG DNA lesions each elicit a signal for the induction of p53 and the induction apoptosis. Taking these tools and extensive """"""""preliminary data"""""""" our specific aims include the following:- further characterize the global transcriptional response of S. cerevisiae to alkylating and other agents; define the molecular signaling events that occur between sensing alkylated DNA bases and executing cell cycle check points and apoptosis; explore structure-function relationships for 3MeA DNA glycosylases, in particular the glycosylases that cause mutator phenotypes. The health relatedness of this project lies in the fact that it will contribute to our understanding of some of the vents that lead to carcinogenesis.
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