| Polycyclic aromatic hydrocarbons (PAHs) are metabolized to highly reactive PAH diol epoxides that can bind covalently to cellular DNA, forming covalent adducts predominantly with guanine and adenine. The conformations of the bulky lesions derived from the binding of PAH compounds to DNA depend on the structure of the PAH, the stereochemistry of the diol epoxide metabolites, and the base sequence context in which the adducts are embedded. The general goals of this work were to determine how these factors influence DNA replication and nucleotide excision repair using site-specifically inserted oligonucleotides containing single, stereochemically defined adducts derived from the binding of the enantiomeric R,S,S,R and S,R,R,S diol epoxides of the bay region benzo[a]pyrene (B[a]P) and the fjord region benzo[c]phenanthrene (B[c]Ph) to the N2-position of guanine. Unlike the two stereoisomeric adducts derived from B[a]P that are positioned on the minor grooves of B-DNA with opposite orientations (Cosman, M. et al., Proc. Natl. Acad. Sci. USA 1992, 89, 1914–1918), the stereochemically analogous B[c]Ph adducts are intercalated from the minor groove on opposite sides of the modified guanine in the same base sequence context.; Nucleotide excision repair assays using UvrABC proteins from Escherichia coli demonstrate that the trans-B[ c]Ph-N2-dG adducts are excised with higher efficiencies in comparison with the minor groove trans-B[ a]P-N2-dG adducts. However, the reasons underlying these differences remain to be investigated.; The characteristics of primer extension in (+)-trans-anti-B[ a]P-N2-dG lesion-modified templates catalyzed by pol kappa and by the large fragment of the polymerase from the thermophilic Bacillus stearothermophilus (BF) were also studied in vitro. In the latter case, primer extension in 5′ -…CG*G…-3′′ and 5′-…TG*G…-3′ (G* = (+)-trans-anti-B[a]P-N 2-dG) templates is error-prone and was found to be temperature- and base sequence-dependent. In the case of pol kappa, error-free bypass of these bulky lesions is dominant, but the efficiencies of primer extension are strongly dependent on base sequence context. |
