STEREOSPECIFICITY OF HUMAN DNA-POLYMERASE-ALPHA, DNA-POLYMERASE-BETA, DNA-POLYMERASE-GAMMA, DNA-POLYMERASE-DELTA AND DNA-POLYMERASE-EPSILON, HIV-REVERSE TRANSCRIPTASE, HSV-1 DNA-POLYMERASE, CALF THYMUS TERMINAL TRANSFERASE AND ESCHERICHIA-COLI DNA-POLYMERASE-I IN RECOGNIZING D-THYMIDINE AND L-THYMIDINE 5'-TRIPHOSPHATE AS SUBSTRATE

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-I DNA polymerase in vitro,but also human DNA polymerases alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase I and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerases beta, gamma, beta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases dearly incorporate at least one L-dTMP residue, with DNA polymerase a 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.