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 x 10(-4) and 1.3 x 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.
Sala
M
M
Unité de Rétrovirologie Moléculaire, Institut Pasteur, Paris, France.
Wain-Hobson
S
S
Schaeffer
F
F
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D054303:HIV Reverse Transcriptase; D007700:Kinetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D009843:Oligoribonucleotides / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014158:Transcription, Genetic
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase