The goal of the research proposed here is to examine the reaction mode of selected chemical carcinogens with regulatory regions of genes and the effect of this modification on gene expression. I will employ the epidermal growth factor receptor promoter sequence and contiguous dG sequence found in the chicken betaA globin gene. In this study, I will directly address the question of whether the DNA structure serves as an important determinant affecting the accessibility of promoter sequences to carcinogens. Promoter regions of active genes are often hypersensitive to DNase 1 and single-stranded DNA-specific Sl nuclease digestion suggesting that they are forming non-Watson-Crick-type (non-B) DNA structures. Many sequences in the promoter/enhancer regions are also found to be reactive with chloroacetaldehyde, a chemical carcinogen which reacts exclusively with unpaired DNA bases, at specific sites of chromatin in cells and remain to be reactive with the chemical in the form of supercoiled plasmid DNA. Thus many eukaryotic regulatory sequences possess inherent potential in forming non-B DNA structures. Our previous work showed that the DNA sites assuming non-B DNA structures are prime targets of various carcinogens such as chloroacetaldehyde, N-acetoxy-2-aminofluorene, and glycidaldehyde in supercoiled DNA. I will study whether selected chemical carcinogens, glycidaldehyde, N-acetoxy-2-acetylaminofluorene, benzo(a)pyrene and its active metabolites, dinitropyrene and its active metabolites, also react with regulatory DNA sequences in vivo and if such modifications affect the regulatory mechanisms of gene expression in cells after replication. The chemically-modified sites both in vitro and in vivo will be determined at a single-base resolution level with our recently-devised chemical cleavage method employing: 1) supercoiled plasmid DNA, and 2) extrachromosomal replicating minichromosomes in cells. Subsequently, the resulting mutations will be analyzed. The long-term objective of the study proposed here is to understand if and how specific interactions of chemical carcinogens and DNA play a role in the initiation of chemical carcinogenesis. Also, the method I will employ to study the reactions of DNA sequences participating in non-B DNA structures with carcinogens may, in the future, prove useful to screen putative environmental mutagens and carcinogens.
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