HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE (T)G-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 rev erse transcriptase (t)G:T mispair formation and base pair stability wa s investigated using DNA and RNA templates with 15 bp matched or misma tched DNA primers. (t)G:T mispair formation during primer elongation w as undetectable on (t)DNA-DNA duplexes but occurred with a frequency o f 10(-4) on matched (t)RNA-DNA duplexes. The frequency increased to 7. 0x10(-4) and 1.3x10(-3) on (t)RNA-DNA duplexes with (t)G:T mismatches located 6 and 9 bp beyond the polymerization site. From K-m values at 37 degrees C, the free energy change upon dissociation (Delta G degree s(37)) of the (t)G:T mispair increased from matched to mismatched (t)R NA-DNA duplexes by 0.3-1.21 kcal/mol. Delta G degrees(37) for a correc t (t)G:C pair decreased by 0.06-1.00 kcal/mol. In comparison with DNA- DNA duplexes, thermal melting measurements on RNA-DNA duplexes demonst rated smaller enthalpy (Delta Delta H degrees = -17.7 to -28.1 kcal/mo l) and entropy (Delta Delta S degrees = -59.3 to -83.4 caI/mol/K) comp onents. 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 stabili ty in solution, the RNA-DNA duplex presents more facile base pair open ing and higher conformational flexibility. The release of helical stra in at constant helix stability in RNA-DNA duplexes may facilitate base mispairing during reverse transcription, particularly in the context of lentiviral G-->A hypermutation.