Using Single Molecule FRET to Study Hepatitis C Virus NS3h and NS3-NS4A Helicase Unwinding Open Access

Nguyen, Ngoc Minh (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/tx31qj266?locale=en
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Abstract

Hepatitis C virus infection results in development of chronic liver diseases. A potential target for drugs to prevent the development of the virus is the NS3 protein that has helicase and primase activities. In the last few years several studies have described mechanistic aspects of the helicase, but the role of the interaction with NS4A cofactor in the NS3 function is not clear. In this paper, we describe the use of fluorescence and single molecule fluorescence resonant energy transfer (FRET) to understand the mechanisms of binding and unwinding of DNA-DNA substrates and RNA-DNA substrates by the NS3 helicase domain and the NS3-NS4A complex. Our results indicate that multiple units bind to single strand substrates with very high affinity and that unwinding of RNA-DNA occurs at faster rates than unwinding of DNA-DNA. Our results provide the basis for more complete and systematic studies that will help determine the mechanisms of these proteins.

Table of Contents

Table of Contents

Page

1. Abstract ................................................................................................................1

2. Introduction ..........................................................................................................1

a. Hepatitis C virus ......................................................................................1

b. Helicases ..................................................................................................2

c. NS3-NS4A ...............................................................................................4

d. Fluorescence Resonance Energy Transfer (FRET)..................................5

e. Single Molecule FRET (smFRET) ..........................................................6

3. Objective ..............................................................................................................7

4. Material and Methods ..........................................................................................7

a. Experimental Setup ..................................................................................7

b. DNA Substrates .......................................................................................9

c. Bulk Solution Experiments ......................................................................10

d. PEG Slide .................................................................................................10

e. Data Analyses .........................................................................................11

5. Results ..................................................................................................................11

a. smFRET Binding Experiments ................................................................11

b. Unwinding Experiments ..........................................................................14

c. Ensemble Experiments.............................................................................14

6. Discussion ............................................................................................................17

7. Acknowledgements ..............................................................................................20

8. References ............................................................................................................21

9. Appendix A: Protocols .........................................................................................24

10. Appendix B: Figures ............................................................................................40

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