Polycyclic aromatic hydrocarbons (PAH) are byproducts of fossil fuel combustion and are present in our air, food and water;the presence of these genotoxic environmental carcinogens in our environment continues to be a hazard to human health. The structural features and biological impact of PAH that distinguish highly active mutagens and tumorigens from structurally related less active, or inactive compounds, have long been of interest for understanding the etiology of human cancers in exposed populations. There are two important sub-classes of PAH that are distinguished by different topological features: (i) the sterically hindered 'fjord'region that causes significant non-planarity in the aromatic ring system of fjord PAH, and (ii) 'bay'region PAH that are sterically unhindered and planar (e.g., the well known environmental carcinogen benzo[a]pyrene). Both types of PAH are metabolically activated to reactive diol epoxide intermediates that react predominantly with guanine and adenine in cellular DNA to form pre-mutagenic covalent adducts in mammalian cells and tissues that can, if not repaired, ultimately contribute to the etiology of human cancers The fjord PAH have attracted significant attention by the chemical carcinogenesis community because they are up to ~ 100-fold more tumorigenic than the bay region prototype benzo[a]pyrene. Previous research from this laboratory has shown that there are remarkable differences in the relative excision efficiencies of different, stereochemically well defined PAH-DNA adducts by the human nucleotide excision repair (NER) system in whole cell extracts. Of particular interest are the observations that PAH- adenine DNA adducts derived from highly tumorigenic fjord PAH are strongly resistant to NER. However, these conclusions are built on NER experiments that were conducted with free DNA in aqueous environments;it is not known whether in biologically more relevant protein environments of nucleosomes, the fundamental sub-units of DNA packaging in the cell, similar hierarchies of NER will be observed with DNA substrates containing the same single bay and fjord PAH-DNA adducts. The objectives of this project are to evaluate NER efficiencies in nucleosomal DNA.
The specific aims are: (1) to investigate the role of DNA positioning sequence, thermodynamic stability, nucleosome dynamics, and accessibility of DNA adducts at different sites of the nucleosomal superhelix on NER efficiencies;(2) Evaluate the effects of covalent modification of H3, H4, H2A and H2B histones on the same nucleosome properties and NER;(3) Determine how structurally distinct fjord and bay region PAH-DNA adducts in nucleosomes are differentially excised by the NER system with human cell extracts in nucleosomes, and elucidate how the excision efficiencies are impacted by the histone environment compared with uncomplexed DNA The outcome of this study is expected to provide a molecular basis for future investigations of how bulky carcinogen-DNA lesions are processed in vivo and how nucleosome remodeling factors enhance and facilitate the NER of bulky DNA lesions in cellular environments.
The findings of this project will provide a novel basis for identifying the most persistent and therefore the most hazardous PAH carcinogen-DNA adducts in human tissues and fluids, as these likely contribute significantly to the etiology of cancer. These advances will stimulate the development of next-generation biomarkers of environmental exposure that will advance cancer prevention approaches. The novel insights to be developed, concerning which structural features of DNA lesions account for their lack of recognition and highest resistance to cellular DNA repair, will facilitate the design of more effective cancer therapeutic agents.
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