Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: Relating biological consequences to conformational preferences

Environmental polycylic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modifications to the bases. The (+)- and (-)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N⁶ position of adenine, forming bulky (+)-1R- or (-)-1S-trans-anti-[BPh]-N⁶-dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N⁶-dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-specific, stereochemically pure [BPh]-N⁶-dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but significant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.