The goals of this proposal are 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 at attempt to relate these structural changes to the mutations that they induce. The data generated in these studies will be used to test the hypothesis that the type of mutation induced is dependent on the adduct structure and the DNA sequence within which it is located. We will also attempt to specifically relate this analysis to the question of the importance of non-targeted or semi-targeted mutagenesis. In addition, these site- specifically modified templates will also be used to characterize the stable complex which we have observed to form between a DNA lesion and a DNA helicase. This proposal is focussed on two important and well- studied classes of carcinogenic DNA adducts: aromatic amines and polycyclic aromatic hydrocarbons. However, our long-term objective is to develop techniques to place any DNA lesion into a specific DNA sequence, even 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 makes the synthesis prohibitive. The relevance of this work towards the progress of understanding the initiation of cancer in eukaryotic cells is two- fold. First, studying the relationship between adduct structure within a specific sequence context and induced mutations and mutation frequency will lead to an understanding of the parameters which define a mutational hotspot, many of which are thought to be important in oncogene activation. Second, studying the irreversible binding of a protein to a DNA adduct site may clarify the role these structures might play in the induction of chromosomal breaks or rearrangements.
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