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Title: Mutagenesis studies of the varicella-zoster virus thymidylate synthase
Author: Harrison, Patrick Thomas
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Abstract:
Varicella-zoster virus (VZV), the causative agent of chickenpox and shingles, encodes approximately 70 different proteins including the enzyme thymidylate synthase (TS). TS catalyses the reductive methylation of deoxyuridylate to form thymidylate using N5,N10-methylene-tetrahydrofolate as a cofactor. The enzyme plays a pivotal role in the provision of an essential precursor for DNA synthesis. As such it has been used as a target for antimetabolites which are effective against a variety of infectious and proliferative diseases. The work presented in this thesis describes the establishment of two random mutagenesis protocols suitable for the generation of complete mutant DNA libraries of the VZV TS gene. Use of these libraries, in conjunction with a novel screening protocol, has shown that a single nucleotide change is not sufficient to give rise to a VZV TS variant resistant to the potent inhibitors 5-fluoro-2'-deoxyuridine-monophosphate (FdUMP) and 5-ethynyl-2'-deoxy-uridine-monophosphate (EYdUMP). This is in contrast to a variant of human TS which contains a single nucleotide substitution that has been proposed to confer resistance to FdUMP. The introduction of the equivalent nucleotide substitution into the VZV TS gene by site-directed mutagenesis (which resulted in the equivalent amino acid change) did not result in a drug resistant viral enzyme. The mutant library was used to isolate variants of VZV TS that lack catalytic activity. Molecular modelling of the effects of the amino acid substitutions that gave rise to these inactive variants, extended our understanding of the plasticity of protein structure. As a complementary approach to investigating the structure and function of TS, site-directed mutagenesis was used to study the effects of amino acid changes in two specific regions of the protein; one buried and one exposed to solvent. In the buried region it was shown that the enzyme is more flexible to amino acid substitutions than previously expected, and the mechanism of accomodation of at least one of the changes was identified. A study of the solvent exposed C-terminal region identified structural and functional differences between VZV TS and Lactobacillus casei TS. The two enzymes responded differently to amino acid changes, and the VZV enzyme relied more heavily on hydrophobic contacts to maintain a functional conformation. Phylogenetic comparisons and molecular modelling suggested that the VZV TS may be unique in this regard. Exploitation of the difference between the VZV TS and the human enzyme may allow the rational design of VZV-specific inhibitors and such compounds may have a role as anti-varicella drugs. It was shown that the expression of the active VZV thymidine kinase (TK) was necessary for the phosphorylation of 5-ethynyl-2'-deoxyuridine to a form that would inhibit TS. This led to the identification of an amino acid essential for VZV TK activity in a region of the enzyme not previously thought to play a role in catalysis. The evolutionary source of the herpesvirus TKs has been hitherto obscure. A comparison of the conserved amino acid regions of herpesvirus TKs with cellular deoxycytidine kinase revealed a significant degree of homology. In addition, the herpesvirus TKs and deoxycytidine kinase share common biochemical properties. Taken together, this led to the proposal that the evolutionary source of the herpesvirus TKs was the cellular deoxycytidine kinase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796774  DOI: Not available
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