of Work: This work was initiated to examine whether transcription by RNA polymerase II may contribute to the high variation observed in certain portions of the HIV genome. To address this question we have developed an in vitro system that allows quantitative evaluation of the fidelity of RNA polymerases including purified T7 RNA polymerase(RNAP) and RNA pol II in human cell extracts during synthesis on a natural template in the presence of all four nucleotides. Recently we have employed this system using a TAA-ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase (T7 RNAP) past an adenine residue adducted at the N6 position with (-)anti-trans- or (+)anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full length run-off transcripts. The extent of bypass was found to be 32 % for the (-)anti-trans-isomer and 18 % for the (+)anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass-synthesis of the (-)anti-trans isomer was elevated 11,000-fold and that of the (+)anti-trans isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, 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, 3- and 6-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell. - Cell Extracts, DNA Polymerases, HIV-1, Mutagenesis, Reverse Transcriptase, RNA, Messenger
