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Author

Dennis Huszar

Date of Award

9-1983

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Dr. Silvia Bacchetti

Abstract

Ribonucleotide reductase catalyzes the first unique step in DNA synthesis by reduction of all four ribonucleotides to the corresponding deoxyribonucleotides. Herpes simplex virus (HSV), which codes for at least three enzymes of DNA metabolism (thymidine kinase, DNA polymerase and DNAase) was found to induce a novel ribonucleotide reductase activity upon infection of mammalian cells. The HSV-2 induced reductase was purified essentially free of the endogenous cellular enzyme and found to differ from the cellular reductase in several of its biochemical properties, most notably in its resistance to allosteric inhibition by dTTP and dATP (Huszar and Bacchetti, 1981). In addition, a rabbit antiserum was prepared (Rl serum) which was capable of specifically immunoprecipitating the HSV-2 induced reductase, thus demonstrating that the induced and cellular enzymes could also be immunologically distinguished (Huszar et al., 1983). Further experiments established that Rl serum cross-reacted with two monoclonal antibodies, both specific for HSV-2 polypeptides of approximately 144,000 and 38,000 daltons, which were capable of either immunoprecipitating the HSV-2 induced reductase (H11 antibodies) or directly neutralizing it in solution (Bg7 antibodies) (Huszar et al., 1983).

These data demonstrate that either one or both of the HSV-2 144,000 and 38,000 dalton polypeptides are associated with viral ribonucleotide reductase activity. Based on the mapping of these polypeptides (Anderson et al., 1981; Docherty et al., 1981; Galloway et al., 1982a), these data also locate the coding sequences for at least a component of the enzyme between .56 - .60 map units on the viral genome within DNA sequences associated with cell transformation. The identification of viral DNA sequences coding for, and of viral polypeptides associated with, the HSV-2 ribonucleotide reductase will facilitate studies on the relevance of the enzyme to viral replication, latency and cell transformation.

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