The objective of this research is to investigate the mechanism of mutagenesis and DNA repair induced by the nitrated pyrenes that are known environmental pollutants and are suspected to pose threats to human health. To accomplish this goal, we have used the tools of organic synthesis and recombinant DNA technology to construct specific DNA fragments and viral genomes that contained, at preselected sites, the major DNA adduct formed by 1-nitropyrene, and 1,6- and 1,8-dinitropyrene. In the past we investigated the mutagenic activity of some of these adducts in Escherichia coli. A major objective of the current application is to employ this technology to construct specific mutagenic hot spot sequences from the p53 gene and study sequence-specificity of mutagenesis in mammalian cells and in extracts from repair-proficient and -deficient human cell lines. Progeny DNA molecules will be isolated, and the mutagenic as well as toxic effects of the adduct will be studied both qualitatively and quantitatively. Another major objective of this proposal is to determine the thermodynamics and kinetics of interaction of the nucleotide excision repair proteins from E. coli and human cells, and compare the rates of incision on site-specific adducts in the same DNA sequence context as employed in the mutagenesis studies. Also, detailed physicochemical studies will be carried out on the modified oligonucleotides to understand the architectural effects of the adducts. A primary goal of this work is to define the relationship between the structure and three-dimensional effects of a lesion in DNA and the mutagenicity and DNA repair that it may induce. Another important aim is to understand the mechanism how the repair proteins, and specifically nucleotide excision repair, protect cells against the toxic and mutagenic properties of these DNA damaging agents.
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