Our long term goal is to understand the biology, biochemistry and genetics of the response of cells to DNA alkylation damage. Alkylating agents represent the most abundant class of chemical DNA damaging agents in our environment and they induce cell death and mutation. Since we are continuously exposed to these chemicals, and since certain alkylating agents are used for chemotherapy, it is important to understand exactly how cells respond these agents. It is becoming increasingly clear that organisms separated by enormous evolutionary distances employ similar proteins to protect against damage relentlessly inflicted upon their DNA, and we now know that bacteria, yeast and human cells induce the expression of specific sets of genes in response to DNA damage. Our studies on the response of E. coli, S. cerevisiae and human cells to DNA alkylation damage have become intertwined and are being executed in an integrated fashion. Much of the 'Proposed experiments are based on our findings that bacterial DNA repair functions can operate in eukaryotic cells, and vice versa, i.e., eukaryotic DNA repair functions can operate in bacterial cells. Specifically, we identified and isolated three eukaryotic genes by virtue of their ability to rescue E. coli form alkylation induced killing or nutation; a 3MeA DNA glycosylase, an 06-MeG DNA methyltransferase, and a gene of unknown function. These genes and their products will be characterized, paying particular attention to the specificities of the DNA repair activities and to the transcriptional regulation of the genes in response to DNA alkylation damage, and other types of DNA damage. In addition, a number of other eukaryotic genes (both yeast and human) whose products prevent alkylation induced killing and mutation will also be isolated and similarly characterized. It is anticipated that characterization of all these genes will not only deepen our understanding of the roles of known DNA repair enzymes in protecting cells against the effects of alkylating agents but will also uncover some hitherto unknown protective mechanisms. The health relatedness of this project lies in the fact that it will contribute to an understanding of some of the events that may lead to carcinogenesis (via somatic mutations and to the
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