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.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 (1-4). The exact mechanism of translocation and nucleic acid strand separation is not known for any helicase. However, unwinding is believed to be a stepwise process that among other things may require interaction between helicase molecules. In this paper we use single turnover unwinding kinetics experiments as well as numerical modeling to investigate the role of subunit interactions during unwinding by the helicase from hepatitis C virus.Hepatitis C virus (HCV) 1 contains a single stranded RNA genome that codes for a polyprotein, which is cleaved into structural and nonstructural (NS) proteins. The NS3 protein of the HCV is both a helicase and a protease. The crystal structure of NS3 shows two loosely connected domains (5). The helicase activity resides on the C-terminal domain that constitutes ϳ450 C-terminal amino acid residues and the protease activity on the N-terminal domain. The NS3 protease is tightly associated with its essential co-factor NS4A, which is predicted to be membrane-bound. The NS3 helicase is, therefore, tethered to the endoplasmic reticulum membrane in vivo. The protease and helicase activities appear to be independent as these domains can be expressed separately in Escheric...

SummaryCspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of b-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA. We propose that CspD dimers, with two independent b-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.