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

EMBO J. 1995 Sep 15;14(18):4622-7.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition
  • Base Sequence
  • DNA / chemistry
  • DNA / genetics*
  • DNA Primers*
  • HIV Reverse Transcriptase
  • Kinetics
  • Molecular Sequence Data
  • Nucleic Acid Denaturation
  • Nucleic Acid Heteroduplexes / chemistry
  • Oligodeoxyribonucleotides / genetics
  • Oligoribonucleotides / genetics
  • RNA / chemistry
  • RNA / genetics*
  • RNA-Directed DNA Polymerase / metabolism*
  • Substrate Specificity
  • Thermodynamics
  • Transcription, Genetic*

Substances

  • DNA Primers
  • Nucleic Acid Heteroduplexes
  • Oligodeoxyribonucleotides
  • Oligoribonucleotides
  • RNA
  • DNA
  • HIV Reverse Transcriptase
  • RNA-Directed DNA Polymerase