A Dimer of Escherichia coli UvrD is the Active Form of the Helicase In Vitro

Maluf, Nasib K.; Fischer, Christopher J.; Lohman, Timothy M.

doi:10.1016/s0022-2836(02)01277-9

Cited by 199 publications

(345 citation statements)

References 30 publications

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“…Such oligomerization could be indicative of a rolling model like the ones proposed to explain the mechanism of action of other helicases such as Rep (46) and UvrD (33,47). Based on an analysis of crystal packing interactions, Cho et al (8) proposed that HCV helicase could form a dimer by forming contacts between helicase domains 1 and 2 of two separate monomers.…”

Section: Effects Of Ns3 Protease On Hcv Helicasementioning

confidence: 94%

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

Frick

Rypma

Lam

et al. 2004

Journal of Biological Chemistry

103

128

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The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3 -5 helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA (or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.The epidemic caused by infection by the hepatitis C virus (HCV) 1 is still a global crisis despite recent therapeutic advancements (1). Because HCV cannot be conventionally cultivated in cell culture and the only other host is the chimpanzee, the enzymes encoded by HCV have been studied intensely as targets for rational drug design. One key viral enzyme is the multifunctional nonstructural protein 3 (NS3), which possesses a serine protease activity and an ATPase function that fuels the ability of the protein to unwind RNA and DNA duplexes. Although it is clear that both the protease and helicase functions are necessary for viral replication (2), it is not clear whether the two functions, which reside in independent protein domains, cooperate in any manner (3).To examine possible effects of the NS3 protease on its helicase function, the activities of the full-length NS3 protein were rigorously compared with the same protein lacking the protease and also with recombinant proteins in which the protease is replaced with other non-HCV peptides. The experiments were designed to uncover effects of the NS3 protease domain on its helicase function which are either specific or nonspecific. Nonspecific effects are defined as those that can be duplicated by peptides not derived from HCV NS3, whereas specific effects cannot.All recombinant proteins used in this study ( Fig. 1) were derived from the same infectious clone of HCV genotype 1a (4). NS3 spans amino acids 1027-1659 of the ϳ3,000-amino acid long polyprotein encoded by HCV. At the N terminus resides the pro...

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Section: Effects Of Ns3 Protease On Hcv Helicasementioning

confidence: 94%

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

Frick

Rypma

Lam

et al. 2004

Journal of Biological Chemistry

103

128

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“…However, a different type of cooperative behavior that does not require protein-protein interactions arises from the mere presence of multiple helicase molecules on the same nucleic acid substrate. The superfamily 1 helicases Rep and UvrD helicases from E. coli have been proposed to function as dimers (22,23), but multiple UvrD helicase molecules on the DNA show increased activity (23)(24)(25). RecQ and PcrA helicases have been proposed to function as monomers (26,27), but it is not known if multiple helicase molecules have an increased activity.…”

mentioning

confidence: 99%

The Functional Interaction of the Hepatitis C Virus Helicase Molecules Is Responsible for Unwinding Processivity

Levin

Wang

Patel

2004

Journal of Biological Chemistry

116

134

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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...

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“…If this ratio falls between 0.25 and 1, then a helicase can be considered as active. Most helicases likely fall between these extremes, and for some SF1 helicases, comparison of unwinding and translocation rates is complicated by oligomerization that occurs during unwinding [119,134,135]. However, bacteriophage T4 Dda has been shown to unwind duplex DNA and translocate on ssDNA at the same rate suggesting that it functions by a completely active mechanism [136].…”

Section: Duplex Dna Is Split By the Pin Regionmentioning

confidence: 99%

“…The nucleic acid unwinding processivity of some SF1 helicases can be increased significantly either through self-assembly or interactions with accessory proteins [119,121,134,135,160]. Rep helicase (SF1A) exists as a monomer in the absence of DNA [161].…”

Section: Protein-protein Interactions That Regulate Helicase Activitymentioning

confidence: 99%

“…However, Rep undergoes a DNA-induced dimerization upon binding either ss or dsDNA [132,162], and the dimer appears to be the active form of the Rep helicase [119,132,161,[163][164][165]. Single turnover kinetic studies of UvrD-catalyzed DNA unwinding suggested that dimers are the minimal oligomeric form needed for optimal helicase activity [121,134,135]. In the case of Pif1, binding of ssDNA induces protein dimerization [166].…”

Section: Protein-protein Interactions That Regulate Helicase Activitymentioning

confidence: 99%

See 1 more Smart Citation

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

Raney

Byrd

Aarattuthodiyil

2012

Advances in Experimental Medicine and Biology

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Superfamily I is a large and diverse group of monomeric and dimeric helicases defined by a set of conserved sequence motifs. Members of this class are involved in essential processes in both DNA and RNA metabolism in all organisms. In addition to conserved amino acid sequences, they also share a common structure containing two RecA-like motifs involved in ATP binding and hydrolysis and nucleic acid binding and unwinding. Unwinding is facilitated by a "pin" structure which serves to split the incoming duplex. This activity has been measured using both ensemble and single-molecule conditions. SF1 helicase activity is modulated through interactions with other proteins.

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A Dimer of Escherichia coli UvrD is the Active Form of the Helicase In Vitro

Cited by 199 publications

References 30 publications

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

The Functional Interaction of the Hepatitis C Virus Helicase Molecules Is Responsible for Unwinding Processivity

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

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