Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function

Abstract

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.