Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Robert D. Kuchta

Second Advisor

Jennifer F. Kugel

Third Advisor

Byron W. Purse

Abstract

Genomic replication is possibly the most crucial process to the perpetuation of any species. For cellular organisms, from E. Coli to Homo sapiens, this involves the copying of DNA by a class of extremely accurate and processive replicative enzymes: polymerases. Many replicative polymerases have error rates of 10-3 to 10-6, misincorporating a nucleotide only once every 1,000 to 1,000,000 incorporation events. It is currently believed that there is very little ΔG° difference between the formation of correct and incorrect base pairs, and that the polymerase must somehow greatly amplify this ΔΔG° to achieve high levels of fidelity. Current views on the matter stem from melting studies of DNA duplexes containing correct or incorrect base pairs at their termini, which only provide an approximation of base pair stability. We have directly measured ΔG° for the incorporation of correct as well as incorrect nucleotides and found ΔΔG° to be large enough to account for error rates as high as 10-5. Our data indicate that the energetics of base pairing can account for high levels of fidelity with little if any ΔΔG° amplification by the polymerase.

The influenza virus, a non-cellular organism, encodes a multi-functional RNA-dependent RNA polymerase that both replicates its RNA genome as well as transcribes viral mRNA. Genomic replication occurs de novo, while transcription requires the polymerase to utilize its 7-methlyguanosine 5’ cap binding and endonuclease functions to “steal” a 5’ capped segment from a host transcript before it can polymerize influenza mRNA directly on to the 3’ end of the stolen segment. A fundamental question of influenza pathology is how the polymerase begins transcribing mRNA at the early stages of influenza infection and switches to replication during the later stages. We have identified the host 7- methylguanosine 5’ cap source as well as the viral polymerase and template as factors that initiate a switch between transcription and replication.

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