Poxviruses are large double-stranded DNA viruses of metazoans and represent a threat to human populations in the form of bioterrorism and emerging infectious diseases. The first steps of poxvirus DNA synthesis yield concatemeric arrays of covalently linked genomes. The virus-encoded Holliday junction resolving enzyme, or resolvase, is required to process concatemers into unit-length genomes for packaging and thus represents an attractive drug target. Poxvirus resolvase is a member of the RNase H-like superfamily of nucleotidyl phosphotransfer enzymes, which include RNase H, integrase, transposase, Holliday junction resolvase, and Argonaute. These enzymes require divalent metal-ions for activity and share a similar αβ-fold in their catalytic domains which brings together three or four conserved acidic residues to bind the metal-ions and form the enzyme active site. Small molecules containing a diketo acid-like moiety are potent inhibitors of HIV integrase and are thought to disrupt catalysis by metal-ion chelation at the active site. These studies suggest similar inhibitors could be found against poxvirus resolvase.
In this work we studied the poxvirus resolvase enzymes encoded for by vaccinia virus and fowlpox virus. We found that the two enzymes display similar DNA cleavage properties, but that the purified vaccinia enzyme was insoluble and not well suited for more detailed analyses. We identified the fowlpox enzyme as a biochemically tractable poxvirus resolvase, which was well suited for basic kinetic analysis, DNA binding studies, and importantly, high throughput screening. We interrogated the DNA binding and cleavage activities of the enzymes using a combination of different DNA substrates, amino acid substitutions, divalent metal-ion conditions, and over 133,000 small molecules. The results suggest that poxvirus resolvase may play a general DNA debranching role during the course of viral replication and that small molecules with diketo acid-like pharmacophores could be developed into selective inhibitors that target resolvase inside poxvirus infected cells.
|Advisor:||Bushman, Frederic D., Isaacs, Stuart|
|School:||University of Pennsylvania|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 70/02, Dissertation Abstracts International|
|Keywords:||Holliday junctions, Poxvirus, RNase H, Resolvase|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be