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.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/284045 |
| Date | January 1999 |
| Creators | Baker, Robert Owen |
| Contributors | Hall, Jennifer D. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| 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. |
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