The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity

Mikhail K. Levin, Yuh Hwa Wang, Smita S. Patel

Research output: Contribution to journalArticle

105 Citations (Scopus)

Abstract

Although helicases participate in virtually every cellular process involving nucleic acids, the details of their mechanism including the role of interaction between the subunits remains unclear. Here we study the unwinding kinetics of the helicase from hepatitis C virus using DNA substrates with a range of tail and duplex lengths. The binding of the helicase to the substrates was characterized by electron microscopy and fluorimetric titrations. Depending on the length of the ssDNA tail, one or more helicase molecules can be loaded on the DNA. Unwinding was measured under single-turnover conditions, and the results show that a monomer is active on short duplexes yet multiple molecules are needed to unwind long duplexes. Thus, increasing the ssDNA tail length increases the unwinding efficiency. The unwinding kinetics was modeled as a stepwise process performed by single or multiple helicase molecules. The model programmed in MATLAB was used for global fitting of the kinetics, yielding values for the rate of unwinding, processivity, cooperativity, step size, and occlusion site. The results indicate that a single hepatitis C virus helicase molecule unwinds DNA with a low processivity. The multiple helicase molecules present on the DNA substrate show functional cooperativity and unwind with greater efficiency, although they bind and release the substrate non-cooperatively, and the ATPase cycle of the helicase molecules is not coordinated. The functional interaction model explains the efficient unwinding by multiple helicases and is generally applicable.

Original languageEnglish (US)
Pages (from-to)26005-26012
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number25
DOIs
StatePublished - Jun 18 2004

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Viruses
Hepacivirus
Molecules
DNA
Substrates
Kinetics
Nucleic Acids
Adenosine Triphosphatases
Electron Microscopy
Titration
Electron microscopy
MATLAB
Monomers

ASJC Scopus subject areas

  • Biochemistry

Cite this

The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. / Levin, Mikhail K.; Wang, Yuh Hwa; Patel, Smita S.

In: Journal of Biological Chemistry, Vol. 279, No. 25, 18.06.2004, p. 26005-26012.

Research output: Contribution to journalArticle

Levin, Mikhail K. ; Wang, Yuh Hwa ; Patel, Smita S. / The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 25. pp. 26005-26012.
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