Studies Concerning The Role Of Polymerase-Associated Exonuclease Activity In The Anti-Herpetic Effect Of 9 - Beta-D-Arabinosyladenine
Date of Award
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
Investigations were conducted into the feasibility of inhibiting proofreading exonuclease activity during replication of Salmonella typhimurium, human (HEp-2) cells and Herpes Simplex Virus (HSV) by administration of the antitumor drug 6-mercaptopurine (6-MP). Combinations of 6-MP and 2-aminopurine (2-AP) produced mutational synergism in the bacterial system but synergistic toxicity or antiviral activity was not observed. Combinations of 6-MP and adenine arabinoside (araA) did not produce synergistic toxicity, due to the ability of araA to partially overcome the purine deprivation effect of 6-MP. However, araA and 6-MP displayed synergistic antiviral activity, indicating that proofreading exonuclease activity may be of physiological importance to Herpes Simplex Virus.HSV-induced DNA polymerase was purified and examined for associated nuclease activity. An exonuclease was found to co-fractionate through three chromatographic steps and this novel activity was partially characterized. Hydrolysis of polynucleotide in the 3' to 5' direction, template-dependent turnover of nucleotide and protection of labelled, double-stranded substrate from degradation by complementary deoxynucleoside triphosphate were demonstrated, consistent with the presence of a proofreading exonuclease. Inhibition of nuclease activity by ribonucleoside 5'-phosphates and the resulting increase in stable nucleotide incorporation was also demonstrated.New kinetic data on the effect of inhibition of HSV DNA polymerase-associated nuclease upon incorporation and hydrolysis of araAMP was obtained. Finally, the synergistic inhibition of polynucleotide synthesis by HSV DNA polymerase was demonstrated using araATP and 6-MPR-P. The 2- to 3-fold synergism produced with purified enzyme in vitro was similar to that produced by 6-MP and araA in HSV undergoing replication in cell culture. It is concluded that HSV DNA polymerase-associated exonuclease moderates the antiviral effect of araA by removing incorporated araAMP residues from 3'-primer termini during viral DNA synthesis.
Frank, Karl Bruce, "Studies Concerning The Role Of Polymerase-Associated Exonuclease Activity In The Anti-Herpetic Effect Of 9 - Beta-D-Arabinosyladenine" (1982). Dissertations from ProQuest. 1266.