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SUMMARYSeveral 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 (Fa-dT) were used as substrates for HeLa DNA polymerase a 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 a. 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 K m and Vma X values for different substrates, indicating that the mutation to PFA r has affected the structure of the virus DNA polymerase. Surprisingly, all three enzymes use Fa-dTTP as substrate for DNA synthesis to an equal but limited extent.

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Investigations on deoxythymidine Kinase show that: (a) 5-ethyl-2′-deoxyuridine-β (EDU-β) binds to about 100-fold weaker (K_m 2.7 × 10^-4) than deoxythymidine (K_m 2.5 × 10^-6) and the rate of its phosphoration (0.7 nmol/mg protein/15 min) is about the half of the deoxythymidine (1.52 nmol/mg protein/15 min); (b) 5-ethyl-2′-deoxyuridine-α (EDU-α), 5-propyl-2′-deoxyuridine (α and β) as well as α and β-5-isopropyl-2′- deoxyuridines do not serve as substrates for this enzyme.

Chemical synthesis of C-4 tritium labeled 1′-(2′-deoxy-β-ᴅ-ribofuranosyl) -5-ethyluracil, also named as 5-ethyl-2′-deoxyuridine, a virostatic compound of therapeutic value, is described. The β-configuration has been derived from its optical rotatory dispersion spectrum, NMR spectrum and from its enzymatic hydrolysis.Incorporation of 5-ethyluracil into DNA of phages, bacteria and of mammalian cells has been already demonstrated. To follow the photochemical behavior of 5-ethyluracil in DNA, valid information in vitro can be expected from experiments with the 5-ethyl-2′-deoxyuridine alone; because we have already shown that contrary to 5-ethyl-, 5-propyl- and 5-butyluracil their N-1 and/or N-3 substituted mono and/or di-alkyl derivatives undergo photodimerisation. Similarly, as expected 5-ethyl-2′-deoxyuridine dimerises upon U.V.-Irradiation also (48%). Under the same experimental conditions thymidine dimerises practically to same extent as well (49%). From these results in vitro it follows that replacement of thymidine with 5-ethyluracil into DNA should not essentially alter its normal behavior towards U.V.-light.