Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase.
The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5'-exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3'-azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate.
Nickel
W
W
Max-Planck-Institut für Experimentelle Medizin, Abetilung Chemie, Göttingen, Germany.
Austermann
S
S
Bialek
G
G
Grosse
F
F
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D019915:DNA Primase; D004261:DNA Replication / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D054306:Dideoxynucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D011067:Poly dA-dT / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism
D054306:Dideoxynucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D013942:Thymine Nucleotides; D011067:Poly dA-dT; D015215:Zidovudine; C052873:zidovudine triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase