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J Biol Chem, Vol. 273, Issue 33, 21276-21281, August 14, 1998

Effects of Nonbulky DNA Base Damages on Escherichia coli RNA Polymerase-mediated Elongation and Promoter Clearance

Anand Viswanathan and Paul W. Doetsch^§

From the  Graduate Program in Genetics and Molecular Biology and ^§ Departments of Biochemistry and Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia 30322

DNA base damage products either formed spontaneously or as a result of exposure to various genotoxic agents were examined for their effects on Escherichia coli RNA polymerase-mediated transcription in vitro. Uracil, O⁶-methylguanine (O⁶-meG), and 8-oxoguanine (8-oxoG) were placed at specific sites downstream from the transcriptional start site on the transcribed strand of a duplex template under the control of the strong tac promoter. In vitro, single-round transcription experiments carried out with purified E. coli RNA polymerase revealed efficient bypass at the three lesions examined and subsequent generation of full-length runoff transcripts. Transcript sequence analysis revealed that E. coli RNA polymerase inserted primarily adenine into the transcript opposite to uracil, uracil opposite to O⁶-meG, and either adenine or cytosine opposite to 8-oxoG. Thus, a uracil in the DNA template resulted in a G-to-A transition mutation in the lesion bypass product whereas O⁶-meG produced a C-to-U transition mutation and 8-oxoG generated either the correct transcriptional product or a C-to-A transversion mutation. When 8-oxoG was placed within close proximity to the transcriptional start site (within the region required for effective promoter clearance), a reduced of full-length, runoff transcript was observed, indicative of lower promoter clearance. Taken together, these results demonstrate that the DNA base damages studied here may exert significant in vivo effects on gene expression and DNA repair with respect to the production of mutant proteins (transcriptional mutagenesis), or decreased levels of expressed proteins.

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