Herpes viruses comprise a large family of complex, double-stranded DNA viruses, a number of which are serious human pathogens. Herpes DNA replication requires a group of virally encoded proteins, and is the target of several antiviral drugs. However, the mechanism of Herpes replication is remarkably complex and not well understood. The broad, long-term goal of these studies is to obtain a detailed biochemical understanding of the mechanisms of Herpes primase and polymerase, and how they function together to initiate the synthesis of new DNA strands. This knowledge will, in turn, substantially enhance our understanding of the mechanism by which Herpes viruses replicate their DNA.
The specific aims of this proposal are: 1. Obtain a detailed understanding of the role of UL8 during primer synthesis. Even though this protein is essential for Herpes replication, little is known about how it functions mechanistically. 2. Develop a detailed understanding of how primase decides whether or not to polymerize a NTP. Primase is an astoundingly inaccurate enzyme, yet it is very capable of discriminating against NTPs containing modified bases. These studies will elucidate the fidelity mechanisms that give rise to these unusual properties 3. Determine how primase interacts with the sugar of a nucleotide and then synthesize potent and specific inhibitors of this enzyme. Insights obtained from the studies on primase fidelity (Aim 2) will be combined with the results of studies on the interaction of primase with sugar analogs to develop novel inhibitors. 4. Elucidate the mechanism of primase-coupled DNA polymerase activity. These studies will determine how primase and polymerase function together to initiate the synthesis of new DNA strands. 5. Determine how Herpes polymerase discriminates between correct and incorrect dNTPs. Fidelity of DNA replication is one of the most fundamental issues facing DNA polymerases. These studies will determine how Herpes polymerase obtains fidelity during DNA replication. To accomplish these aims, steady state and pre-steady state kinetic approaches, photoactivateable crosslinking reagents, and DNA footprinting methodologies will be used. Additionally, a large number of novel nucleotides will be synthesized.
|Dickerson, Sarah Michelle; Kuchta, Robert D (2017) Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 during Helicase-Coupled DNA Synthesis by the Herpes Polymerase. Biochemistry 56:2651-2662|
|Vashishtha, Ashwani Kumar; Kuchta, Robert D (2016) Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA. Biochemistry 55:1168-77|
|Vashishtha, Ashwani Kumar; Kuchta, Robert D (2015) Polymerase and exonuclease activities in herpes simplex virus type 1 DNA polymerase are not highly coordinated. Biochemistry 54:240-9|
|Rodgers, Brittney J; Elsharif, Nada A; Vashisht, Nisha et al. (2014) Functionalized tricyclic cytosine analogues provide nucleoside fluorophores with improved photophysical properties and a range of solvent sensitivities. Chemistry 20:2010-5|
|Weller, Sandra K; Kuchta, Robert D (2013) The DNA helicase-primase complex as a target for herpes viral infection. Expert Opin Ther Targets 17:1119-32|
|Olson, Andrew C; Patro, Jennifer N; Urban, Milan et al. (2013) The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication. J Am Chem Soc 135:1205-8|
|Lund, Travis J; Cavanaugh, Nisha A; Joubert, Nicolas et al. (2011) B family DNA polymerases asymmetrically recognize pyrimidines and purines. Biochemistry 50:7243-50|
|Stengel, Gudrun; Kuchta, Robert D (2011) Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates. J Virol 85:957-67|
|Stengel, Gudrun; Purse, Byron W; Kuchta, Robert D (2011) Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs. Anal Biochem 416:53-60|
|Chen, Yan; Bai, Ping; Mackay, Shannon et al. (2011) Herpes simplex virus type 1 helicase-primase: DNA binding and consequent protein oligomerization and primase activation. J Virol 85:968-78|
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