Transcription-coupled repair (TCR), a sub-pathway of the nucleotide  excision repair (NER), removes lesions from the template strands of  expressed genes after initiation induced by RNA polymerase stalling.   Various bulky lesions have been shown to cause transcription arrest, it  is, however, still unclear why some lesions strongly block RNA  polymerase while others do not.  The focus of this study was to evaluate  the role of altered dNTP/rNTP base-pairing in transcription elongation.   In vitro T7 RNA polymerase transcription assays were performed  using DNA templates containing purine analogues with altered H-bonding  capacities.  The results show that C2-amination of purine blocks RNA  synthesis.  In contrast, the oxidative lesion 2-hydroxy-dA is  effectively bypassed, revealing a previously unknown role of a C2-amino  group in polymerase stalling.  Transcriptional efficiency was  furthermore independent of DNA duplex stability, with the  duplex-destabilizing purine and a G-T mismatch being effectively  bypassed by the polymerase.  The results are presented in context of T7  RNAP substrate selection, and suggest that disfavored hydrogen-bonding  in the closed active site of RNA polymerase terminates transcription  elongation.

Altered DNA minor groove hydrogen-bonding as a trigger for T7 RNA polymerase transcriptional stalling

244th National Meeting (2012)

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