Analysis of primary sequence data revealed a subset of open reading frames that were predicted to encode HSV-I dUTPases based on five areas of local primary sequence conservation. The differences in the primary sequence organisation of these motif regions allowed the description of two distinct dUTPase classes. The class I dUTPases are encoded by a diverse range of organisms and are characterised by a trimeric arrangement with subunit protein lengths approximating 150 amino acids. The class II dUTPases are specific to the herpesviruses and are characterised by a monomeric arrangement with a protein chain length approximately double that of their class I counterparts. It has been proposed that the class II dUTPases arose by the intragenic duplication of the class I open reading frame. In this thesis the class I structures were used as a basis to investigate the HSV-1 class II dUTPase in terms of structural and evolutionary relationships. To allow a defined approach to functional analysis of the HSV-1 dUTPase a tertiary structural model was generated for the class II enzymes. Following intensive primary sequence analysis a method was devised for comparing class I and class II sequences directly. Secondary structure prediction programs were utilised to judge the basic structural similarities between the two classes allowing the proposition of several defined hypotheses. The available class I structural information was utilised in order to characterise highly conserved structural elements within the class I group. In was then possible to relate this data set to class I primary sequences and subsequently to the generation of a class II model. Various modelling techniques were used based on the constraints on the structural organisation that could achieve a functionally active monomer plus the set of hypotheses defined in the earlier work. Mutagenic analysis of the HSV-1 dUTPase was then possible using the class II model as a reference. Several targets were investigated based on predicated functionally important regions. Analysis of these mutant enzymes was performed using purified recombinant HSV-1 dUTPase expressed from the T7 E.coli expression system.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:284931 |
| Date | January 1998 |
| Creators | McGeehan, John Edward |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/6104/ |
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