DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAP) and modifications in the template DNA may stall transcription and initiate biochemical defense mechanisms, including transcription-coupled repair of DNA. The major DNA modifications found to stall transcription are bulky adducts that distort DNA geometry and hinder the polymerase active site, thus impairing ribonucleoside incorporation. Some non-bulky DNA adducts, however, were also observed to stall transcription, while others did not. For example, transcription appears to stall at O6-methylguanine, but not at 5-hydroxycytosine or thymidine diols. Considering that a nucleobase modification may alter their H-bonding abilities, we became interested in assessing the impact that dNTP:rNTP base-pair geometry and DNA:RNA heteroduplex stability may have on transcription over non-bulky DNA adducts. The obtained results demonstrated that DNA:RNA duplex destabilization upon mismatch formation does not appear to contribute to transcriptional stall unless caused by wobble pairs. Thus, transcription by a bacteriophage T7 RNA polymerase was stalled at wobble dNTP:rNTP pairs and not at non-H-bonded mismatches, suggesting that RNA polymerase can discriminate between mismatch types. Furthermore, the stalling of RNA polymerase at wobble base-pairs appears to depend on the complementarity of a non-transcribed DNA strand, revealing a novel role of a non-coding strand in proofreading of RNA synthesis and, putatively, in substrate recognition by transcription-coupled repair.

Chemistry of DNA stability and transcription

248th National Meeting (2014)

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