Deoxyuridine in DNA has an inhibitory and promutagenic effect on RNA transcription by diverse RNA polymerases.

dUTP is a close structural congener of dTTP and can be readily incorporated into DNA opposite to adenine during DNA replication leading to non-mutagenic dU/A base pairs ('uracilation'). We find that dU/A pairs located within DNA transcriptional templates optimized for either T7 RNA polymerase (T7 RNAP) or human RNA polymerase II (pol II) have inhibitory and mutagenic effects on transcription. The data for T7 RNAP establishes that even a single dU/A pair can inhibit promoter binding and transcription initiation up to 30-fold, and that inhibitory effects on transcription elongation are also possible. Sequencing of the mRNA transcribed from uniformly uracilated DNA templates by T7 RNAP indicated an increased frequency of transversion and insertion mutations compared to all T/A templates. Strong effects of dU/A pairs on cellular transcription activity and fidelity were also observed with RNA pol II using uracil base excision repair (UBER)-deficient human cells. At the highest levels of template uracilation, transcription by RNA pol II was completely blocked. We propose that these effects arise from the decreased thermodynamic stability and increased dynamics of dU/A pairs in DNA. The potential implications of these findings on gene regulation and disease are discussed.