Effects of primer-template sequence on ATP-dependent removal of chain-terminating nucleotide analogues by HIV-1 reverse transcriptase

J Biol Chem. 2004 Oct 29;279(44):45389-98. doi: 10.1074/jbc.M405072200. Epub 2004 Aug 11.


HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3'-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2',3'-dideoxy-AMP, 2',3'-dideoxy-CMP, or 2',3'-dideoxy-GMP. However, the removal of 2',3'-dideoxy-TMP or 3'-azido-2',3'-dideoxy-TMP was much less influenced by upstream primer-template sequence, and the rate of excision of these thymidylate analogues was greater than or equal to that of the other chain-terminating residues in each sequence context tested. These results strongly indicate that the primer terminus and adjacent upstream base pairs interact with reverse transcriptase in a sequence-dependent manner that affects the removal reaction. We conclude that primer-template sequence context is a major factor to consider when evaluating the removal of different chain terminators by HIV-1 reverse transcriptase.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Base Sequence
  • DNA Primers / chemistry*
  • Deoxyadenine Nucleotides / metabolism*
  • Dideoxynucleotides
  • HIV Reverse Transcriptase / metabolism*
  • Molecular Sequence Data
  • Templates, Genetic*


  • DNA Primers
  • Deoxyadenine Nucleotides
  • Dideoxynucleotides
  • 2',3'-dideoxyadenosine 5'-phosphate
  • Adenosine Triphosphate
  • HIV Reverse Transcriptase