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.
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.
Focher
F
F
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Maga
G
G
Bendiscioli
A
A
Capobianco
M
M
Colonna
F
F
Garbesi
A
A
Spadari
S
S
D000818:Animals; D002417:Cattle; D004253:DNA Nucleotidylexotransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006367:HeLa Cells / Q000201:enzymology; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D011071:Poly T / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors; D013237:Stereoisomerism; D013379:Substrate Specificity; D013936:Thymidine / Q000737:chemistry / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000737:chemistry / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D011071:Poly T; D013938:Thymidine Monophosphate; D013937:Thymidine Kinase; D004253:DNA Nucleotidylexotransferase; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C024157:thymidine 5'-triphosphate; D013936:Thymidine