Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide

Biochemistry. 1989 Feb 7;28(3):1340-6. doi: 10.1021/bi00429a060.


Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).

Publication types

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

MeSH terms

  • Binding, Competitive
  • DNA Replication
  • HIV / enzymology*
  • Kinetics
  • Mathematics
  • Nucleic Acid Synthesis Inhibitors
  • Oligodeoxyribonucleotides / pharmacology*
  • RNA-Directed DNA Polymerase / metabolism*
  • Reverse Transcriptase Inhibitors
  • Templates, Genetic
  • Thionucleotides / pharmacology*


  • Nucleic Acid Synthesis Inhibitors
  • Oligodeoxyribonucleotides
  • Reverse Transcriptase Inhibitors
  • Thionucleotides
  • Sd(C)28
  • RNA-Directed DNA Polymerase