INCORRECT BASE INSERTION AND PREMATURELY TERMINATED TRANSCRIPTS DUPING T7 RNA-POLYMERASE TRANSCRIPTION ELONGATION PAST BENZO[A]PYRENEDIOL EPOXIDE-MODIFIED DNA

DNA replication and transcription are affected adversely by the presen ce of bulky adducts that are generated by the covalent binding of a va riety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes pri or to replication, mutations and ultimately tumor initiation can occur . Transcription and DNA repair appear to be intimately connected, sinc e certain adducts are more efficiently removed from the transcribed st rands of active loci than from non-transcribed strands and other quies cent domains in the genome. The mechanism by which RNA polymerases dea l with bulky adducts during DNA transcription is therefore of great in terest. The availability of site-specifically modified and stereochemi cally defined oligodeoxyribonucleotides derived from the covalent reac tion of 7r, 8t-dihydroxy-9, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e (anti-BPDE) with guanine residues prompted us to study the efficienc ies of transcription past these lesions using bacteriophage T7 RNA pol ymerase. We show here that T7 RNA polymerase can bypass such lesions i n a DNA template, providing that a cytosine residue is incorporated op posite anti-BPDE-modified guanine. However, when an incorrect base (mo st frequently a purine) is inserted opposite the modified site, the RN A polymerase stalls, and the complex dissociates, resulting in a trunc ated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mec hanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template. (C) 1996 Acade mic Press Limited