EFFECTS OF NONBULKY DNA-BASE DAMAGES ON ESCHERICHIA-COLI RNA POLYMERASE-MEDIATED ELONGATION AND PROMOTER CLEARANCE

DNA base damage products either formed spontaneously or as a result of exposure to various genotoxic agents were examined for their effects on Escherichia coil RNA polymerase-mediated transcription in vitro, Ur acil, O-6-methylguanine (O-6-meG), and 8-oxoguanine (8-oxoG) were plac ed at specific sites downstream from the transcriptional start site on the transcribed strand of a duplex template under the control of the strong too promoter. In vitro, single-round transcription experiments carried out with purified E. coil RNA polymerase revealed efficient by pass at the three lesions examined and subsequent generation of full-l ength runoff transcripts. Transcript sequence analysis revealed that E , coil RNA polymerase inserted primarily adenine into the transcript o pposite to uracil, uracil opposite to O-6-meG, and either adenine or c ytosine opposite to 8-oxoG. Thus, a uracil in the DNA template resulte d in a G-to-A transition mutation in the lesion bypass product whereas O-6-meG: produced a C-to-U transition mutation and 8-oxoG; generated either the correct transcriptional product or a C-to-A transversion mu tation. When 8-oxoG was placed within close proximity to the transcrip tional start site (within the region required for effective promoter c learance), a reduced of full-length, runoff transcript was observed, i ndicative of lower promoter clearance. Taken together, these results d emonstrate that the DNA base damages studied here may exert significan t in vivo effects on gene expression and DNA repair with respect to th e production of mutant proteins (transcriptional mutagenesis), or decr eased levels of expressed proteins.