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Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity.

An Li1, Shanzhong Gong1, Kenneth A Johnson2

  • 1From the University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712.

The Journal of Biological Chemistry
|October 26, 2016
PubMed
Summary

Pyrophosphate release is the rate-limiting step in RNA-templated DNA synthesis, enhancing enzyme fidelity. This slow pyrophosphate release significantly improves discrimination against incorrect nucleotide incorporation.

Keywords:
DNA polymeraseDNA replicationHIV-1 reverse transcriptaseenzyme kineticsglobal data fittinghuman immunodeficiency virus (HIV)presteady-state kineticspyrophosphate releasetransient kinetics

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Biochemistry

Background:

  • Previous studies indicated rapid pyrophosphate release did not limit enzyme specificity in DNA-templated synthesis.
  • Understanding nucleotide incorporation and pyrophosphate release kinetics is crucial for elucidating DNA polymerase fidelity.

Purpose of the Study:

  • To determine kinetic parameters of nucleotide incorporation and pyrophosphate release using an RNA template.
  • To investigate the role of pyrophosphate release in DNA polymerase fidelity during reverse transcription.

Main Methods:

  • Kinetic analysis of nucleotide incorporation and pyrophosphate release.
  • Comparison of reaction rates using DNA and RNA templates.
  • Determination of rate constants for correct and incorrect nucleotide incorporation.

Main Results:

  • RNA-templated synthesis showed a 10-fold increase in chemistry rate but significantly slower pyrophosphate release (58 s⁻¹), becoming rate-limiting.
  • Sequential nucleotide incorporation rates were limited by slow pyrophosphate release.
  • Pyrophosphate release was extremely slow during misincorporation (∼0.002 s⁻¹), enhancing discrimination by over 20-fold compared to previous estimates.

Conclusions:

  • Slow pyrophosphate release is the rate-determining step in RNA-templated nucleotide incorporation.
  • This slow release mechanism significantly contributes to DNA polymerase fidelity during reverse transcription.
  • Revised discrimination estimates, including slow pyrophosphate release, reveal a >20-fold increase in polymerase accuracy.