Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate

Nucleic Acids Res. 1995 Aug 11;23(15):2840-7. doi: 10.1093/nar/23.15.2840.


L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • DNA Nucleotidylexotransferase / antagonists & inhibitors
  • DNA Nucleotidylexotransferase / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / enzymology
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism
  • HIV Reverse Transcriptase
  • HIV-1 / enzymology*
  • HeLa Cells / enzymology
  • Herpesvirus 1, Human / enzymology
  • Humans
  • Nucleic Acid Synthesis Inhibitors
  • Poly T / metabolism
  • RNA-Directed DNA Polymerase / metabolism*
  • Reverse Transcriptase Inhibitors
  • Stereoisomerism
  • Substrate Specificity
  • Thymidine / chemistry
  • Thymidine / metabolism
  • Thymidine Kinase / genetics
  • Thymidine Kinase / metabolism
  • Thymidine Monophosphate / chemistry
  • Thymidine Monophosphate / metabolism
  • Thymine Nucleotides / chemistry
  • Thymine Nucleotides / metabolism*
  • Thymine Nucleotides / pharmacology


  • Nucleic Acid Synthesis Inhibitors
  • Reverse Transcriptase Inhibitors
  • Thymine Nucleotides
  • Poly T
  • Thymidine Monophosphate
  • Thymidine Kinase
  • DNA Nucleotidylexotransferase
  • HIV Reverse Transcriptase
  • RNA-Directed DNA Polymerase
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V
  • thymidine 5'-triphosphate
  • Thymidine