Title

A repertoire of transactions in the herpes simplex virus type-1 genome

Date of Award

2004

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Paul E. Boehmer, Committee Chair

Abstract

Herpes simplex virus type-1 is the causative agent of diseases including oropharyngeal lesions, herpetic encephalitis and corneal blindness. HSV-1 lends itself as an amenable eukaryotic based system to study genomic transactions including DNA synthesis. HSV-1 is a relatively large (152 kbp) enveloped double-stranded DNA virus which has been well characterized biochemically and genetically. The work presented in the fulfillment of this thesis explores a myriad of processes that relate the involvement of the initiator protein, the single-strand DNA binding protein and heat shock proteins at viral origins of replication, as well as the participation of the viral polymerase in translesion synthesis.Firstly, we examined the role of cysteines in the mechanism of action of UL9. We mutagenized conserved cysteines to alanine and found that cysteine 111 is involved in coupling single-stranded DNA binding to ATP binding and subsequent hydrolysis.A second set of studies examined the mechanism by which ICP8 stimulates UL9. Because both the DNA helicase and DNA-stimulated ATPase activities of UL9 are enhanced greatly by its association with ICP8, we speculated that the stimulatory effect was attributed to the capacity of ICP8 to tether UL9 to template DNA. The data presented herein shows that, contrary to this model, ICP8's single-strand DNA binding activity is dispensable for its stimulation of UL9. Instead, ICP8 sequesters an inhibitory domain of UL9, resulting in its stimulation.Previous observations from our laboratory suggest a role for heat shock proteins in activating UL9 to initiate efficient viral origin-dependent DNA replication. Our goal was to correlate the in vitro findings with an in vivo system. Preliminary data shows that Hsp70 and Hsp40 can be up-regulated but as of yet, the effect does not correlate with enhanced origin-dependent replication.Lastly, we examined the role of the replicative polymerase, UL30, in translesion synthesis. A long-standing view has been that polymerases with exonuclease activity engage in a futile cycle of incorporation and excision (idling) at sites of DNA damage, because any incorporation is viewed by the polymerase as a misincorporation event. Therefore, we examined the role of exonuclease activity in the ability of UL30 to perform translesion synthesis.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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