Analysis of the DNA Binding Domain of the Herpes Simplex Virus Type 1 UL9 Protein

Arbuckle, Margaret Isabel (1993) Analysis of the DNA Binding Domain of the Herpes Simplex Virus Type 1 UL9 Protein. PhD thesis, University of Glasgow.

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The UL9 gene is one of seven genes which map within the long unique region of the HSV-1 genome and are both necessary and sufficient for viral origin-dependent DNA replication in transfected tissue culture cells. The UL9 gene encodes a polypepetide of 851 amino acids which binds specifically to the HSV-1 replication origins ori3 and oriL. From DNase I footprinting and gel retardation analyses, two binding sites for the origin binding protein (OBP) have been identified within both of these origins. Previous work (Weir et al., 1989) demonstrated that the sequence-specific recognition and binding activities resided within the C-terminal 317 amino acids of the UL9 protein. My work has involved a mutational analysis of the UL9 DNA binding domain in an attempt to define the regions involved in interaction with its target sequence. Using an E.coli expression system, a series of C- terminal deletion (with 16 - 62 amino acids deleted) and small in-frame insertion mutants of the DNA binding domain were constructed and expressed as fusions linked to the N-terminal one third of the Staphylococcus aureus protein A. This system enabled easy detection of mutant proteins in Western blot assays by virtue of interaction of the Fc portions of the antibody conjugates with the protein A moieties. Fusions proteins were tested for sequence-specific DNA binding activity using gel retardation assays. Protein extracts were incubated with radio-labelled oligonucleotides containing ori3 binding sites I or II or a 100 bp fragment containing a functional ori3 (i.e. both sites) at 22

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Nigel D Stow
Keywords: Virology
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-76368
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 15:20
Last Modified: 19 Nov 2019 15:20

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