This program project seeks to define the molecular basis for structure- activity relationships among PAHs which leads some to be potent carcinogens and others inactive. Efforts are focused on the DNA adducts of the diol epoxide metabolites. The program has two overriding goals. The first is to discover the structural determinants for an adduct to be intercalated versus lying in the groove, i.e., how the arrangement of PAH rings and configurations of PAH functional groups determines the conformation of ds DNA. The second major goal is to discover the conformational determinants for an adduct to be mutagenic versus non-mutagenic. Current results from this program reveal that site-specifically placed PAH adducts vary widely in their effects on efficiency and fidelity of polymerase bypass. Three laboratories have joined forces to address these goals. Under the direction of Dr. T.M. Harris (Vanderbilt Univ.) flexible and efficient methodology is being developed for preparation of oligonucleotides containing adducts of a wide variety of PAH diol epoxides. The syntheses are designed such that both the location of the adduct and the absolute configurations of PAH functional groups are rigorously defined. Highlights of Dr. Harris's project include development of an enantioselective synthesis of PAH diol epoxides and a post-oligomerization approach to the adducted oligonucleotides. The adducted oligonucleotides are being used by Dr. R. S. Lloyd (UTMB - Galveston) to establish by in vitro experiments the effects on polymerase passage and by in vivo experiments the mutagenic spectra an frequencies in bacterial and human cells. The effect of adducts on polymerase action has been found to be crucially dependent on the absolute configuration of PAH carbon atom at the site of attachment and whether the adduct is derived from a bay region or non-bay region diol epoxide. Structural studies by Dr. M. P. Stone (Vanderbilt Univ.) are being used to interpret the biochemical and biological results. Multidimensional NMR spectroscopy is the main experimental tool for examination of solution structure of the adducted oligonucleotides in normal and mismatched duplexes and for additional experiments involving DNA-polymerases complexes. Other structural tools to be used include fluorescence measurements and x-ray crystallography. Present results show that the PAH structure and absolute configuration of the attachment site fundamentally affect duplex conformation. Proposed studies will seek to rationalize mutational events.
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