To accurately replicate its viral genome, the Herpes Simplex Virus 1 (HSV-1) DNA polymerase usually polymerizes the correct natural 2′-deoxy-5′-triphosphate (dNTP) opposite the template base being replicated. We employed a series of purine-dNTP analogues to determine the chemical features of the base necessary for the herpes polymerase to avoid polymerizing incorrect dNTPs. The enzyme uses N-3 to prevent misincorporation of purine dNTPs but does not require N-3 for correct polymerization. A free pair of electrons on N-1 also helps prevent misincorporation opposite A, C, and G and strongly drives polymerization opposite T. N 6 contributes a small amount both for preventing misincorporation and for correct polymerization. Within the context of guanine in either the incoming dNTP or the template base being replicated, N 2 prevents misincorporation opposite adenine but plays at most a minor role for incorporation opposite C. In contrast, adding N 2 to the dNTPs of either adenine, purine, 6-chloropurine, or 1-deazapurine greatly enhances incorporation opposite C, likely via the formation of a hydrogen bond between N 2 of the purine and O2 of the pyrimidine. Herpes polymerase is very sensitive to the structure of the base pair at the primer 3′-terminus since eliminating N-1, N-3, or N 6 from a purine nucleotide at the primer 3′-terminus interfered with polymerization of the next two dNTPs. The biological and evolutionary implications of these data are discussed.
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