Stable complexes formed by HIV-1 reverse transcriptase at distinct positions on the primer-template controlled by binding deoxynucleoside triphosphates or foscarnet

J Mol Biol. 2007 May 25;369(1):41-54. doi: 10.1016/j.jmb.2007.03.006. Epub 2007 Mar 12.

Abstract

Binding of the next complementary dNTP by the binary complex containing HIV-1 reverse transcriptase (RT) and primer-template induces conformational changes that have been implicated in catalytic function of RT. We have used DNase I footprinting, gel electrophoretic mobility shift, and exonuclease protection assays to characterize the interactions between HIV-1 RT and chain-terminated primer-template in the absence and presence of various ligands. Distinguishable stable complexes were formed in the presence of foscarnet (an analog of pyrophosphate), the dNTP complementary to the first (+1) templating nucleotide or the dNTP complementary to the second (+2) templating nucleotide. The position of HIV-1 RT on the primer-template in each of these complexes is different. RT is located upstream in the foscarnet complex, relative to the +1 complex, and downstream in the +2 complex. These results suggest that HIV-1 RT can translocate along the primer-template in the absence of phosphodiester bond formation. The ability to form a specific foscarnet complex might explain the inhibitory properties of this compound. The ability to recognize the second templating nucleotide has implications for nucleotide misincorporation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Footprinting
  • DNA Primers / metabolism*
  • DNA, Complementary / metabolism
  • Deoxyribonuclease I / metabolism
  • Exodeoxyribonucleases / metabolism
  • Foscarnet / metabolism*
  • HIV Reverse Transcriptase / metabolism*
  • Nucleotides / metabolism*
  • Protein Binding
  • Templates, Genetic*

Substances

  • DNA Primers
  • DNA, Complementary
  • Nucleotides
  • Foscarnet
  • HIV Reverse Transcriptase
  • Exodeoxyribonucleases
  • Deoxyribonuclease I