Thermodynamics of A:G mismatch poly(dG) synthesis by human immunodeficiency virus 1 reverse transcriptase

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase has been found to conduct error-prone synthesis on DNA and RNA templates. We find here that tolerance of an A:G mispair with poly(rA) as template is particularly strong, such that extensive poly(dG) synthesis is conducted. This type of extensive misincorporation is not observed with several reference DNA polymerases. Surprisingly, HIV reverse transcriptase processivity and kcat for dGMP misincorporation and normal dTMP incorporation are about the same. However, the Km value for dGTP in poly(dG) synthesis is approximately 1000-fold higher than the Km for dTTP in poly(dT) synthesis. Comparison of thermodynamic parameters for dGMP misincorporation and normal dNMP incorporation indicates a lower energy of activation for dGMP misincorporation than for normal dNMP incorporation. Entropy of activation (delta S*) for normal dTMP incorporation is positive (approximately 10 cal/kmol), whereas delta S* for dGMP misincorporation is negative (-36 cal/kmol). Since differences in delta S* are usually considered to reflect differences in solvation for the transition state complex, these results are consistent with the interpretation that the active site of HIV reverse transcriptase is flexible enough to misincorporate dGMP without the usual dispersion of water molecules.