The molecular motor nonstructural protein 3 (NS3) is an essential protein expressed by the hepatitis C virus (HCV) known to catalyze the unwinding of both DNA and RNA substrates. We investigated the role of binding in the unwinding mechanism by determining the outcome of the enzyme's interaction with nucleic acid in the absence of ATP binding and hydrolysis. Comparing full-length NS3 to the helicase domain (NS3h) revealed fundamental mechanistic differences in the way these enzymes interact with substrates due largely to the fact that NS3 is capable of forming large oligomeric structures, whereas NS3h functions as a dimer. The NS3h dimer was confirmed via footprinting methods and gel filtration. NS3h is capable of unwinding long partial-duplex substrates in the absence of a nucleotide cofactor, and our data support the existence of an underlying ATP-independent directionally-biased sliding mechanism. Our data suggest that 3' to 5' movement on ssNA is an intrinsic property of the helicase that occurs without requiring hydrolysis of a nucleotide cofactor. In contrast, NS3 utilizes its oligomeric property to bind directly to the duplex region of partial-duplex substrates and locally separate base pairs in an ATP-independent fashion. This action is limited to duplex lengths at or near the kinetic step size of 18 bp. Additionally, there appears to be two modes of unwinding utilized by full-length NS3: a translocation-independent mode by the action of the large oligomeric species and a translocation-dependent mode likely due to the actions of a smaller species (i.e. dimer).
|Advisor:||Raney, Kevin D.|
|Commitee:||Baldini, Giulia, Fifer, Espero K., Miller, Paul G., Tackett, Alan|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 74/09(E), Dissertation Abstracts International|
|Keywords:||DNA unwinding, Directionally-based sliding, Helicase, Hepatitis C virus|
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