The long-term goal of this project is to provide insight into the molecular mechanisms by which carcinogens may effect tumor formation through genetic changes following their alteration of DNA. Experimentally, this objective is being pursued by the construction of infectious DNA molecules that carry single, site-specific carcinogen adducts. Introduction of these adducts into prokaryotic and eukaryotic cells allows one to specifically relate the change in DNA sequence of the progeny to the presence of a structurally defined adduct in the parent molecule. Differences in adduct structure will be related to the biochemical, molecular biological and biological consequences of these DNA adducts with the intent of elucidating and characterizing those pathways that are crucial to the genotoxic effects of these agents. A key objective is to determine whether the mutagenic effects of these compounds come from their ability to trigger a cellular response that is independent of adduct structure and/or whether different adduct structures have different mutagenic potentials because they perturb the conformation of the replication complex at the time of DNA synthesis.
The specific aims of these studies are to aid in clarifying the following questions: 1) What are the structural features that determine the mutagenic response to C8 aromatic amine adducts with guanine? 2) Are different mechanisms of repair and/or mutagenicity responsible for the response to these structures? 3) Are similar responses elicited in prokaryotic and eukaryotic cells, are they produced by similar mechanisms? Synthetic oligonucleotides that contain a single guanine will be modified with reactive aromatic amines to introduce C8 adducts at a specific base. Initially, 2-aminofluorene and 4-aminobiphenyl derivatives, with or without N-acetyl groups, will be studied; additional candidates that complement the structures of these planar and nonplanar ring systems will be evaluated by use of molecular modelling techniques prior to their synthesis. The adducts will be ligated into gapped heteroduplex DNA molecules that have single stranded regions that are precisely complementary to the oligonucleotide. DNA adducts that have uracil in the strand complementary to the adduct will also be used to enhance the mutagenic response. The adducts will be introduced into cells with different genetic backgrounds to better define the mechanisms responsible for the generation of adduct- dependent mutations. The progeny of the adducts will be screened for changes in nucleotide sequence by oligonucleotide hybridization, a technique that permits the identification of mutated sequences without a selection bias. The actual mutational events will be determined by direct sequencing. In vitro experiments will provide indirect information on conformational differences of the oligonucleotides, as reflected in the ability of enzymes to degrade them or to use them as templates for polynucleotide synthesis.
