ERA

Download the full-sized PDF of Vaccinia virus DNA polymerase and ribonucleotide reductase: their role in replication, recombination and drug resistanceDownload the full-sized PDF

Analytics

Share

Permanent link (DOI): https://doi.org/10.7939/R3QD1W

Download

Export to: EndNote  |  Zotero  |  Mendeley

Communities

This file is in the following communities:

Graduate Studies and Research, Faculty of

Collections

This file is in the following collections:

Theses and Dissertations

Vaccinia virus DNA polymerase and ribonucleotide reductase: their role in replication, recombination and drug resistance Open Access

Descriptions

Other title
Subject/Keyword
recombination
DNA polymerase
vaccinia virus
ribonucleotide reductase
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Gammon, Donald Brad
Supervisor and department
Evans, David (Medical Microbiology and Immunology)
Examining committee member and department
Reha-Krantz, Linda (Biological Sciences)
Barry, Michele (Medical Microbiology and Immunology)
Condit, Richard (Molecular Genetics and Microbiology, University of Florida)
Schang, Luis (Biochemistry)
Department
Department of Medical Microbiology and Immunology
Specialization

Date accepted
2009-12-16T21:04:15Z
Graduation date
2010-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Despite the eradication of smallpox, poxviruses continue to cause human disease around the world. At the core of poxvirus replication is the efficient and accurate synthesis and repair of the viral genome. The viral DNA polymerase is critical for these processes. Acyclic nucleoside phosphonate (ANP) compounds that target the viral polymerase are effective inhibitors of poxvirus replication and pathogenesis. Cidofovir (CDV) is an ANP that inhibits vaccinia virus (VAC) DNA polymerase (E9) DNA synthesis and 3’-to-5’ exonuclease (proofreading) activities. We determined that point mutations in the DNA polymerase genes of ANP-resistant (ANPR) VAC strains were responsible for CDV resistance and resistance to the related compound, HPMPDAP. Although these resistant strains replicated as well as wild-type VAC in culture, they were highly attenuated in mice. The generation of ANPR VAC strains, in combination with our knowledge of how CDV inhibits E9 activities, allowed us to study the hypothesized role of E9 in catalyzing double-strand break repair through homologous recombination. We provide evidence that VAC uses E9 proofreading activity to catalyze genetic recombination through single-strand annealing reactions (SSA) in infected cells. Both the polarity of end resection of recombinant intermediates and the involvement of polymerase proofreading activity establish these poxviral SSA reactions as unique among homologous recombination schemes. Furthermore, we identified roles for the VAC single-stranded DNA-binding (SSB) protein and nucleotide pools in regulating these reactions. During these later studies we uncovered a differential requirement for the large and small subunits of the VAC ribonucleotide reductase (RR) in viral replication and pathogenesis. Our studies suggest that poxviral RR small subunits form functional complexes with host large RR subunits to provide sufficient nucleotide pools to support DNA replication. We present a model whereby interaction of VAC SSB and RR proteins at replication forks allows for modulation of E9 activity through local nucleotide pool changes, which serves to maximize replication rates while still allowing for recombinational repair.
Language
English
DOI
doi:10.7939/R3QD1W
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication

File Details

Date Uploaded
Date Modified
2014-05-01T00:05:03.654+00:00
Audit Status
Audits have not yet been run on this file.
Characterization
File format: pdf (Portable Document Format)
Mime type: application/pdf
File size: 5901077
Last modified: 2015:10:12 13:52:48-06:00
Filename: Gammon_Donald_Spring 2010.pdf
Original checksum: bd289df01c5c92b362e4c296c090682d
Well formed: true
Valid: false
Status message: Invalid page tree node offset=5898132
Status message: Invalid outline dictionary item offset=5760525
Activity of users you follow
User Activity Date