Tautomeric and anionic Watson-Crick-like mismatches play important roles in replication and translation errors through mechanisms that are not fully understood. Using NMR relaxation dispersion, we resolved a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches consisting of two rapidly exchanging tautomeric species (Genol•T/U⇌G•Tenol/Uenol; population <0.4%) and one anionic species (G•T−/U−; population ≈0.001% at neutral pH). Inserting the sequence-dependent tautomerization/ionization step into a minimal kinetic mechanism for correct incorporation during replication following initial nucleotide binding leads to accurate predictions of dG•dT misincorporation probability across different polymerases, pH conditions, and for a chemically modified nucleotide, and provides mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization/ionization accounts for ≈10−2−10−3–fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol with contributions from anionic dG•dT− dominating at pH ≥8.4 or for some mutagenic nucleotides.

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