This proposal studies the mechanisms by which cells respond to DNA damaging agents. A specialized technology developed early in this project will be used to define the relationships among the structures of lesions formed in the genome by carcinogens and the biological endpoints of mutation, cancer and lethality. This proposal addresses our longstanding interest on the genotoxic mechanisms of three classes of agents: simple DNA alkylating agents, a furanocoumarin (aflatoxin) epoxide, and the reactive species associated with inflammation. By studying a carefully chosen range of DNA lesion structures, we seek to understand the strategies used in biology to address the challenge of DNA damage. While the work principally focuses on challenges to cellular DNA repair and DNA replication systems, we also propose here a new dimension to the project through an attempt to understand the challenges faced by damage to RNA nucleotides. Some of our work probes the biochemistry of enzymes that act upon damage in vitro, but the signature element of our program is its focus on using chemistry and genetics to understand in vivo mechanisms. Most of the work begins with the construction of intact viral or plasmid vectors containing, at one genome site, one of the DNA lesions hypothesized to be responsible for mutagenesis or toxicity. Following introduction of the site-specifically modified genome into bacterial or mammalian cells, the genome is replicated either intra- or extra-chromosomally. We have the ability to control exposure to repair enzymes and, to a certain extent, the polymerases that encounter the lesion in vivo. The type, amount and genetic requirements for mutagenesis and lesion lethality are evaluated. By comparison of lethality and mutagenesis in different cell lines, it is possible to determine to what extent specific genetic backgrounds protect, or sensitize, the cell to specific DNA or RNA lesions.
DNA damage can kill cells and, if the cell survives, causes mutations that could engender cancer or other genetic diseases. This proposal uses chemical tools to construct genomes of viruses that contain the DNA lesions, or adducts, formed by carcinogens. The work then progresses to a genetic phase that helps establish rules that predict the types of DNA damage that give rise to the kinds of mutations observed in genetic diseases.
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