Many environmental and industrial chemicals, found to be mutagenic/carcinogenic when metabolized, form exocyclic derivatives of nucleic acid bases. The chemicals in this category to be studied include two metabolites of benzene, 2-hydroxy-p-benzoquinone and Z,Z-muconaldehyde, and glycidaldehyde, a carcinogenic metabolite from the widely used glycidol ethers. These metabolites will all react with DNA bases, resulting in structures which have additional 5 or 6-membered ring(s) between the 1, N6 of A, the 3, N4 of C and the 1, N2 of G. These adducts, when incorporated into DNA, will be studied in terms of resulting changes in nucleic acid conformation using thermodynamics, molecular modeling and NMR. Concurrently, their mutagenic potential will be assessed by measuring the extent and type of base incorporation opposite the adducts and subsequent lesion bypass in replication using both prokaryotic and eukaryotic DNA polymerases. Experimentally, we will synthesize modified deoxynucleosides and their phosphoramidites which will be site-specifically incorporated into defined oligomers with differing flanking sequences since it is known that the sequence context is an important variable in biochemical functions. For replication studies, a primer extension assay with a 32P-label will be followed by gel electrophoresis for analysis of mispairing. Structural studies will be carried out using thermodynamics, NMR and molecular modeling. Such approaches should yield information on the relationship of adduct structure to DNA duplex stability and local conformational alterations, as well as probable biological relevance of these adducts on the basis of in vitro mutagenic findings. By using these exocyclic adducts, which have incremental changes in chemical structure, information will be gained on how structure affects function.
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