This project will determine solution conformations of PAHs adducted to DNA oligomers, using high field NMR spectroscopy, augmented by other biophysical techniques, including electrophoretic mobility studies, fluorescence methods, and crystallography. These will be examined in the context of the human N-ras protooncogene sequences at codons 12 and 61. These were chosen because carcinogenesis induced by PAH is correlated with adduct formation in these sequences, leading to point mutations which result in oncogene activation.
The specific aims are threefold. First, we will probe for a structural basis to explain specific PAH-induced mutations in these sequences, as observed by R.S. Lloyd. We will examine structurally-based hypotheses which may explain site-specific A yields to G mutations for both adenyl N6 10S(61,2)-benzo[a]pyrene and bay region benz[a]anthracene anti-trans adducts in the ras61 sequence. Second, we will attempt to establish linkage between PAH covalent structure and adduct conformation in DNA. Structure-activity relationships will be determined for a series of PAH adducts located site-specifically in the coding regions of ras12 and ras61. We will determine what structural features of PAH dictate propensity toward intercalation. Studies with T.M. and C.M. Harris will involve both bay- and non-bay-region benz[a]anthracenes, which have different mutagenic responses, as revealed by Lloyd. We will examine a series of 10R and 10S-anti-trans-benzo[a]pyrene adducts at guanine N2 in ras12 to determine whether the 10S(12,2) adduct possesses unusual structural properties derived from steric hindrance of the 5-neighbor guanosine, as predicted by the Geacintov group. An attempt will be made to crystallize PAH-abducted oligomers using sticky ended molecules. Third, to understand how PAH adduct conformation modulates the biochemical processing of adducts, a multifaceted approach will be employed to extend structural studies to complexes with DNA polymerases. Two NMR approaches will be used to examine primer-template complexes. In the first, transferred NOEs between incoming mononucleotides and primer-template complexes will be monitored. In the second, primer-template complexes comprised of protein subfragments will be used. NMR will be supplemented with time-resolved fluorescence using the adduct as a fluorescent probe o ascertain adduct- induced changes at the active site of the complex. An attempt will be made to co-crystallize a PAH-adducted primer-template with polymerase II.
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