Bacteriophage T7 RNA polymerase transcription elongation is inhibited by site-specific, stereospecific benzo[c]phenanthrene diol epoxide DNA lesions

Benzo[c]phennthrene diol epoxide (B[c]PhDE), the ultimate carcinogenic meta bolite of the environmental pollutant benzo[c]phenanthrene: reacts with DNA primarily at the exocyclic amino groups of purines, forming B[c]PhDE-DNA a dducts that differ in their stereochemical configurations and their effect on biological processes such as transcription. To determine the effect of t hese stereoisomers on RNA synthesis, in vitro T7 RNA polymerase transcripti on assays were performed using DNA templates modified on the transcribed st rand by either a site-specific (+)-trans- or (-)-trans-anti-B [c]PhDE-N-6-d A lesion located within the sequence 5'-CTCTCACTTCC-3'. The results show th at both (-)-trans-anti-B[c]PhBE-N-6-dA and (+)-trans-anti-B[c]PhDE-N-6-dA b lock RNA synthesis, Furthermore, both B[c]PhDE-dA stereoisomeric adducts le ad to lower levels of initiation of transcription relative to that observed using an unmodified DNA template. In contrast to these results, placement of the adduct on the nontranscribed strand within the template does not imp ede transcription elongation. In addition to the assessment of the effect o f the lesions on transcription elongation, the resulting transcripts were c haracterized in terms of their base composition. A high level of base misin corporation is detected at the 3'-ends of truncated transcripts, with guano sine being most frequently incorporated opposite the modified nucleotide ra ther than the expected uridine, This result supports the notion that transl ocation past a modified base in a DNA template relies in part on correct ba se incorporation, and suggests that stalling of RNA polymerases at damaged sites in DNA may well be dependent on both the presence of the lesion and t he base which is incorporated opposite the modified nucleotide.