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

R. B. Roth, S. Amin, N. E. Geacintov, D. A. Scicchitano

Research output: Contribution to journalArticle


Benzo[c]phenanthrene diol epoxide (B[c]PhDE), the ultimate carcinogenic metabolite of the environmental pollutant benzo[c]phenanthrene, reacts with DNA primarily at the exocyclic amino groups of purines, forming B[c]PhDE-DNA adducts that differ in their stereochemical configurations and their effect on biological processes such as transcription. To determine the effect of these stereoisomers on RNA synthesis, in vitro T7 RNA polymerase transcription assays were performed using DNA templates modified on the transcribed strand by either a site-specific (+)-trans- or (-)-trans-anti-B[c]PhDE-N6-dA lesion located within the sequence 5′-CTCTCACTTCC-3′. The results show that both (-)-trans-antiB[c]PhDE-N6-dA and (+)-trans-anti-B[c]PhDE-N6-dA block RNA synthesis. Furthermore, both B[c]PhDE-dA stereoisomeric adducts lead 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 impede transcription elongation. In addition to the assessment of the effect of the lesions on transcription elongation, the resulting transcripts were characterized in terms of their base composition. A high level of base misincorporation is detected at the 3′-ends of truncated transcripts, with guanosine being most frequently incorporated opposite the modified nucleotide rather than the expected uridine. This result supports the notion that translocation past a modified base in a DNA template relies in part on correct base 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 the base which is incorporated opposite the modified nucleotide.

Original languageEnglish (US)
Pages (from-to)5200-5207
Number of pages8
Issue number17
StatePublished - May 1 2001


ASJC Scopus subject areas

  • Biochemistry

Cite this