Studies Concerning The Role Of Polymerase-Associated Exonuclease Activity In The Anti-Herpetic Effect Of 9 - Beta-D-Arabinosyladenine

Date of Award




Degree Name

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.


Biology, Microbiology

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