Cellular factors involved with, and fidelity of herpes simplex virus replication

Persistent Link:
http://hdl.handle.net/10150/284045
Title:
Cellular factors involved with, and fidelity of herpes simplex virus replication
Author:
Baker, Robert Owen
Issue Date:
1999
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Herpes Simplex Virus type 1 is an important organism not only because it is a member of a family of disease-causing organisms, but it also serves as a model organism for the study of eukaryotic DNA replication. Here I use HSV-1 to investigate two aspects of DNA replication: initiation and proofreading. Cellular factors have been shown to be involved in DNA replication, and especially in initiation, in a variety of viral systems. Previous studies have identified the first cellular factor implicated in initiation of HSV-1 replication, OF-1. In this study, I have purified OF-1 and investigated its composition, binding properties and interactions with the viral origin binding protein UL9. I show that OF-1 is composed of two subunits, one of which contains DNA binding activity. I also found that OF-1 binds specifically to both single- and double-stranded origin DNA, that OF-1 binds most tightly to single-stranded DNA, and that OF-1 shows a preference for which strand is bound. I have demonstrated that, in the presence of UL9, OF-1 exhibits a higher affinity for its target DNA and that OF-1 inhibits the ATPase activity of UL9. I propose that UL9 binds to the origin of replication, loads OF-1 to the origin, and then is displaced by OF-1. Further implications for this model are discussed. I go on to investigate several aspects of error control in the wild type and a 3'-5' proofreading exonuclease mutant DNA polymerase from HSV-1. Proofreading is a primary factor influencing the fidelity of DNA replication. Previous studies in our lab have shown that exonuclease deficient polymerases are incapable of supporting viral growth in vivo. In these studies, I have expressed and purified both wild type and mutant polymerases and investigated their biochemical properties as well as the mechanism of lethality of the mutant. I have found that the mutant polymerase exhibits substantially elevated rates of nucleotide misincorporation as compared to the wild type. In addition, the mutant polymerase is seen to stall at a misincorporation, exhibiting a reduced ability to replicate past a mismatch. Based on these findings, I suggest that the inability of the mutant polymerase to replicate past a misinsertion is the primary cause of the reduced viability of viruses carrying the mutant enzyme.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Molecular and Cellular Biology
Degree Grantor:
University of Arizona
Advisor:
Hall, Jennifer D.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleCellular factors involved with, and fidelity of herpes simplex virus replicationen_US
dc.creatorBaker, Robert Owenen_US
dc.contributor.authorBaker, Robert Owenen_US
dc.date.issued1999en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractHerpes Simplex Virus type 1 is an important organism not only because it is a member of a family of disease-causing organisms, but it also serves as a model organism for the study of eukaryotic DNA replication. Here I use HSV-1 to investigate two aspects of DNA replication: initiation and proofreading. Cellular factors have been shown to be involved in DNA replication, and especially in initiation, in a variety of viral systems. Previous studies have identified the first cellular factor implicated in initiation of HSV-1 replication, OF-1. In this study, I have purified OF-1 and investigated its composition, binding properties and interactions with the viral origin binding protein UL9. I show that OF-1 is composed of two subunits, one of which contains DNA binding activity. I also found that OF-1 binds specifically to both single- and double-stranded origin DNA, that OF-1 binds most tightly to single-stranded DNA, and that OF-1 shows a preference for which strand is bound. I have demonstrated that, in the presence of UL9, OF-1 exhibits a higher affinity for its target DNA and that OF-1 inhibits the ATPase activity of UL9. I propose that UL9 binds to the origin of replication, loads OF-1 to the origin, and then is displaced by OF-1. Further implications for this model are discussed. I go on to investigate several aspects of error control in the wild type and a 3'-5' proofreading exonuclease mutant DNA polymerase from HSV-1. Proofreading is a primary factor influencing the fidelity of DNA replication. Previous studies in our lab have shown that exonuclease deficient polymerases are incapable of supporting viral growth in vivo. In these studies, I have expressed and purified both wild type and mutant polymerases and investigated their biochemical properties as well as the mechanism of lethality of the mutant. I have found that the mutant polymerase exhibits substantially elevated rates of nucleotide misincorporation as compared to the wild type. In addition, the mutant polymerase is seen to stall at a misincorporation, exhibiting a reduced ability to replicate past a mismatch. Based on these findings, I suggest that the inability of the mutant polymerase to replicate past a misinsertion is the primary cause of the reduced viability of viruses carrying the mutant enzyme.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMolecular and Cellular Biologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHall, Jennifer D.en_US
dc.identifier.proquest9960240en_US
dc.identifier.bibrecord.b4027214xen_US
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