Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase

Abstract

The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression. Moreover, after the time taken by an unimpeded enzyme to go from promoter to terminator, the rate of RNA synthesis remained elevated and the number of complete RNA molecules (7000 nucleotides) continued to increase for some time. These results suggest that, if the E. coli RNA polymerase is stopped by an AP site, most often, after a pause, the enzyme resumes elongation of the RNA chain which is continuous over the AP site. Sometimes however, RNA synthesis is definitively interrupted during the pause; the probability of interruption has been estimated to be 0.3 in our experimental conditions. When a nick is placed 5′ to the AP site by an AP endonuclease, the results are similar: most often, the RNA chain is synthesized without interruption past the nick in the template strand. The pause of the E. coli RNA polymerase at this combined lesion appears to be shorter than when the AP site is intact. To investigate whether a nucleotide is placed in the RNA chain in front of the AP site in the template strand by E. coli RNA polymerase, RNA synthesis was taken to completion before using this RNA for protein synthesis and measuring the activity of gene-1 product, T7 RNA polymerase. The result suggests that, after pausing, the E. coli RNA polymerase places a nucleotide in the RNA chain when passing over an AP site. The mechanism of the delayed lethality of T7 coliphages treated with monofunctional alkylating agents, which is due to the appearance of AP sites, is discussed.