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, the investigator has 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. A major objective of this application is to employ this technology to construct specific mutagenic hot spot sequences in DNA. Sequence-specificity of mutagenesis will be investigated by introducing these modified viral genomes in Escherichia coli cells, where the DNA replication and repair systems will act upon the single DNA adduct. Progeny DNA molecules will be isolated, and the mutagenic as well a toxic effects of the adduct will be studied both qualitatively and quantitatively. Replication of the genome in hosts with defined defects in DNA repair will elucidate the roles of different repair systems in mutagenesis/genotoxicity induced by these DNA adducts. Another major objective of this proposal is to determine the thermodynamics and kinetics of interaction of the Uvr proteins, and rates of incision by the UvrABC system on site-specific 1-nitropyrene adducts in the same DNA sequence context as employed in the mutagenesis studies. Also, detailed physico-chemical studies will be carried out on the modified oligonucleotides to understand the architectural effects of the 1-nitropyrene-DNA 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 proteins, 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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