Differential incorporation of thymidylate analogues into DNA by DNA polymerase alpha and by DNA polymerases specified by two herpes simplex viruses.
Several triphosphates (TP) of 5-substituted deoxyuridine (dU), like 5-ethyl (Et), 5-n-propyl (n-Pr), 5-iso-propyl (iso-Pr), 5-n-hexyl (n-Hx), and 5-trifluorothymidine (F3-dT) were used as substrates for HeLa DNA polymerase alpha and for two herpes simplex virus (HSV)-coded DNA polymerases isolated from HeLa cells infected with HSV-1, strain C42 (wild-type), or its mutant resistant to phosphonoformate (PFAr). All polymerases were purified up to the DNA-cellulose column step and they showed comparable specific activities. The incorporation into DNA studied with all the alkyl analogues of dUTP is several times higher with the virus enzymes than with DNA polymerase alpha. The DNA polymerase of the mutant virus incorporates dUTP analogues to a lower extent than the wild-type polymerase. The two virus enzymes also differ in the Km and Vmax values for different substrates, indicating that the mutation to PFAr has affected the structure of the virus DNA polymerase. Surprisingly, all three enzymes use F3-dTTP as substrate for DNA synthesis to an equal but limited extent.
D004247:DNA / Q000096:biosynthesis*; D004257:DNA Polymerase II / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D018139:Simplexvirus / Q000201:enzymology*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism*; D014271:Trifluridine / Q000378:metabolism
D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; C036000:5-isopropyl-2'-deoxyuridine triphosphate; C036001:5-n-hexyl-2'-deoxyuridine triphosphate; C035799:5-ethyl-2'-deoxyuridine triphosphate; D004247:DNA; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate; D014271:Trifluridine