Differential Incorporation of Thymidylate Analogues into DNA by DNA Polymerase   and by DNA Polymerases Specified by Two Herpes Simplex Viruses

Abstract

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.

Differential Substrate Specificity of DNA Polymerase beta and of a DNA Polymerase Induced by Herpes Simplex Virus Type 2 towards Thymidine Triphosphate Analogues

“…1). With EtdUTP as substrate, incorporation of HSV-1 (H1) are included (Kowalzick et al, 1982). The values are the average of 10 determinations comparable to those shown in Fig.…”

“…The assay conditions used for the HSV-2-specified DNA polymerase were the same as previously employed by us for DNA polymerase coded for by HSV-1 (Kowalzick et al, 1982), except that the specific activity of the labelled compounds, [3H]dGTP or [3H]dTTP, was increased to 1200 ct/min/pmol. The assay procedure was performed as published previously (Kowalzick et al, 1982), except that DNA polymerase specified by HSV-2 was present at 0.15 U per 0.15 ml assay and 40 p.l aliquots were taken. In parallel, we assayed HSV-1 polymerase with this experimental protocol and found results similar to those previously published (Kowalzick et al, 1982).…”

“…The assay procedure was performed as published previously (Kowalzick et al, 1982), except that DNA polymerase specified by HSV-2 was present at 0.15 U per 0.15 ml assay and 40 p.l aliquots were taken. In parallel, we assayed HSV-1 polymerase with this experimental protocol and found results similar to those previously published (Kowalzick et al, 1982). Assays were modified by variation of the content of individual nucleoside triphosphates as indicated in the text below and in the figure legends.…”

“…Each value is the average of 15 determinations. The assay conditions for the ~-polymerase and the HSV-l-induced enzyme have been published (Kowalzick et al, 1982). The difference between the two viral enzymes is statistically significant (probability of error lower than 1 ~).…”

“…Several 5-alkylated analogues of thymidylate are readily incorporated into DNA by DNA polymerases 0022-1317/84/0000-5894 $02.00© 1984 SGM specified by different strains of HSV-1, and to a much lower extent by DNA polymerase c~ from HeLa ceils and by DNA polymerases c~ and fl from calf thymus Kowalzick et al, 1982;Sfigi et al, 1980). We report here on the differential incorporation of several dTTP analogues into DNA by a purified DNA polymerase isolated from HeLa cells infected with HSV-2 strain 75 and by the purified DNA polymerase fl of the host cell.…”