Human immunodeficiency virus type 1 reverse transcriptase tG:T mispair formation on RNA and DNA templates with mismatched primers: a kinetic and thermodynamic study.

The relationship between human immunodeficiency virus (HIV) type 1 reverse transcriptase tG:T mispair formation and base pair stability was investigated using DNA and RNA templates with 15 bp matched or mismatched DNA primers. tG:T mispair formation during primer elongation was undetectable on tDNA‐DNA duplexes but occurred with a frequency of 10(‐4) on matched tRNA‐DNA duplexes. The frequency increased to 7.0 × 10(‐4) and 1.3 × 10(‐3) on tRNA‐DNA duplexes with tG:T mismatches located 6 and 9 bp beyond the polymerization site. From Km values at 37 degrees C, the free energy change upon dissociation (delta G degrees 37) of the tG:T mispair increased from matched to mismatched tRNA‐DNA duplexes by 0.36–1.21 kcal/mol. delta G degrees 37 for a correct tG:C pair decreased by 0.06–1.00 kcal/mol. In comparison with DNA‐DNA duplexes, thermal melting measurements on RNA‐DNA duplexes demonstrated smaller enthalpy (delta delta H degrees = −17.7 to −28.1 kcal/mol) and entropy (delta delta S degrees = −59.3 to −83.4 cal/mol/K) components. A strong entropy‐enthalpy compensation resulted in small free energy differences (delta delta G degrees 37 = 0.8 to −2.2 kcal/mol). Thus, although DNA‐DNA and RNA‐DNA duplexes are of comparable stability in solution, the RNA‐DNA duplex presents more facile base pair opening and higher conformational flexibility. The release of helical strain at constant helix stability in RNA‐DNA duplexes may facilitate base mispairing during reverse transcription, particularly in the context of lentiviral G–>A hypermutation.