The objective of this proposal is to continue the development of a general method for the preparation of oligodeoxyribonucleotides containing chemically well-defined damage at unique and specific locations, to use these molecules to determine how specific adducts affect the three-dimensional structure of a DNA duplex, and to attempt to relate these structural changes to the induced mutations. This proposal is focussed on three important, and well- studied carcinogenic DNA adducts-aminofluorene, acetyl aminofluorene, and benzol(a)pyrene-because of the chemical stability of these particular structures. However our long-term objective is to develop the techniques to place any DNA lesion into a specific DNA sequence, including those where the chemistry needed to prepare the lesion is not now known or where the instability of the lesion to currently available oligonucleotide synthesis technology is prohibitive. Two approaches are currently being used to prepare the site-specifically modified oligonucleotides: nucleotide-specific reactions of derivatives of these carcinogens with pre-formed oligonucleotides, and the total chemical synthesis of oligonucleotides using adduct-containing nucleotide precursors. We will use these modified oligonucleotides and a variety of spectral and enzymatic techniques to determine the effect these adducts have on three-dimensional structure of a DNA duplex. Utilizing the techniques we have developed during the past project period, we will position several modified oligonucleotides into gapped heteroduplexes to form site-specifically modified, biologically active DNA molecules. After extensively characterizing these products, they will be introduced into E. coli cells and the induced mutations determined by DNA sequencing of the progeny.
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