HIGHLY MUTAGENIC BYPASS SYNTHESIS BY T7 RNA-POLYMERASE OF SITE-SPECIFIC BENZO[A]PYRENE DIOL EPOXIDE-ADDUCTED TEMPLATE DNA

We have previously developed an in vitro system that allows quantitati ve evaluation of the fidelity of transcription during synthesis on a n atural template in the presence of all four nucleotides. Here, we have employed this system using a TAA ochre codon reversion assay to exami ne the fidelity of transcription by T7 RNA polymerase past an adenine residue adducted at the N-6-position with (-)-anti-trans- or (+)-anti- trans-benzo[a]pyrene diol epoxide (BPDE), T7 RNAP was capable of trans cribing past either BPDE isomer to generate full-length run-off transc ripts, The extent of bypass was found to be 32% for the (-)-anti-trans -isomer and 18% for the (C)-anti-trans-isomer, Transcription past both adducts was highly mutagenic. The reversion frequency of bypass synth esis of the (-)-anti-trans-isomer was elevated 11,000-fold and that of the (+)-anti-trans-isomer 6000-fold, relative to the reversion freque ncy of transcription on unadducted template. Adenine was misinserted p referentially, followed by guanine, opposite the adenine adducted with either BPDE isomer, Although base substitution errors were by far the most frequent mutation on the adducted template, three- and six-base deletions were also observed. These results suggest that transcription al errors, particularly with regard to damage bypass, may contribute t o the mutational burden of the cell.