Transcription elongation past O⁶-methylguanine by human RNA polymerase II and bacteriophage T7 RNA polymerase

O⁶-Methylguanine (O⁶-meG) is a major mutagenic, carcinogenic and cytotoxic DNA adduct produced by various endogenous and exogenous methylating agents. We report the results of transcription past a site-specifically modified O⁶-meG DNA template by bacteriophage T7 RNA polymerase and human RNA polymerase II. These data show that O⁶-meG partially blocks T7 RNA polymerase and human RNA polymerase II elongation. In both cases, the sequences of the truncated transcripts indicate that both polymerases stop precisely at the damaged site without nucleotide incorporation opposite the lesion, while extensive misincorporation of uracil is observed in the full-length RNA. For both polymerases, computer models suggest that bypass occurs only when O⁶-meG adopts an anti conformation around its glycosidic bond, with the methyl group in the proximal orientation; in contrast, blockage requires the methyl group to adopt a distal conformation. Furthermore, the selection of cytosine and uracil partners opposite O⁶-meG is rationalized with modeled hydrogen-bonding patterns that agree with experimentally observed O⁶-meG:C and O⁶-meG:U pairing schemes. Thus, in vitro, O⁶-meG contributes substantially to transcriptional mutagenesis. In addition, the partial blockage of RNA polymerase II suggests that transcription-coupled DNA repair could play an auxiliary role in the clearance of this lesion.