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.
Highlights
Helicases are motor proteins that translocate along DNA or RNA using ATP hydrolysis
Electron Microscopy (EM) of NS3h1⁄7ss/dsDNA Complexes— EM was used to determine the interactions of NS3 protein with a C-terminal His tag (NS3h) with the ssDNA and duplex DNA regions of the partial duplex helicase substrate
The proposed functional interaction model is the simplest model that explains why multiple Hepatitis C virus (HCV) helicase molecules are more efficient in unwinding a long stretch of duplex DNA
Summary
Helicases are motor proteins that translocate along DNA or RNA using ATP hydrolysis. The translocation activity is required for strand separation of the duplex nucleic acids, the elimination of secondary structure in RNA, and to dissociate proteins bound to the nucleic acids [1,2,3,4]. We have previously [17] characterized the helicase activity of NS3h under multiple turnover conditions using a partially duplex DNA substrate that contained a 10-nt-long ssDNA tail and a 33-bp-long duplex. We observed that the unwinding rate decreased sharply upon the addition of small amounts of an ATPase-deficient NS3h mutant, but the amplitude of unwinding changed little This led us to conclude that many NS3h molecules participate in the unwinding of each DNA substrate. It is not clear, whether a single molecule of the HCV helicase can unwind DNA and, in cases where multiple helicase molecules are required, whether they work cooperatively. Paradoxically it appears that multiple HCV helicase molecules are necessary for efficient DNA unwinding
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