The long term goal of these studies is to develop a detailed understanding of the mechanism by which herpes viruses replicate their DNA. This process requires a series of herpes encoded proteins and may also involve cellular proteins. The proposed studies will focus on two complexes containing 5 essential proteins - the herpes polymerase complex (UL30/UL42) and the primase-helicase complex (UL5/UL52/UL8).
The specific aims of the proposal are: (1) Determine if the cellular pol 1 is required for herpes replication using both siRNA knockdown of pol 1 and a specific small molecule inhibitor of pol 1. (2) Develop a mechanistic understanding of how the interplay between polymerase activity, exonuclease activity, and UL42 enhances the fidelity of replication using a variety of kinetic and mechanistic probes (3) Elucidate the mechanistic consequences of coupled activity between the polymerase and helicase and if the helicase can displace tightly bound proteins from DNA. Both steady- state and pre-steady state kinetic approaches will be used. (4) Using purified proteins and a minicircle template that gives efficient leading and lagging strand, examine the mechanistic coupling of the leading and lagging strand replication apparatus and the effects of bound proteins on the progression of the replication fork. These studies will provide novel insights into how the different proteins functionally interact with each other and the mechanism of herpes DNA replication.

Public Health Relevance

Herpes viruses are significant human pathogens and cause a number of potentially very severe diseases. The herpes replication apparatus is the primary target of currently used anti- herpes drugs. The proposed studies will lead to a better understanding of herpes replication, and thus could lead to the development of new and more effective drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI059764-06A1
Application #
8050733
Study Section
Special Emphasis Panel (ZRG1-IDM-R (02))
Program Officer
Challberg, Mark D
Project Start
2011-07-05
Project End
2015-06-30
Budget Start
2011-07-05
Budget End
2012-06-30
Support Year
6
Fiscal Year
2011
Total Cost
$300,571
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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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