DNA Helicases Displace Streptavidin from Biotin-Labeled Oligonucleotides

Morris, Patrick D.; Raney, Kevin D.

doi:10.1021/bi9822269

Cited by 129 publications

(140 citation statements)

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“…Similarly, it reveals that multiple helicase molecules occupy a longer stretch of DNA during unwinding and prevent reannealing of the DNA behind the helicase. Other helicases such as E. coli UvrD helicase (23,24) show similar behavior during unwinding, and Dda helicase shows similar behavior for streptavidin displacement activity (29). In UvrD helicase both the amplitude and unwinding rate increase with increasing ssDNA tail length.…”

Section: Resultsmentioning

confidence: 93%

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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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Section: Resultsmentioning

confidence: 93%

“…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. Dda helicase functions as a monomer during DNA unwinding (28) but appears to have optimal streptavidin displacement activity as an oligomer (29).…”

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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“…Many helicases have been shown to move unidirectionally along nucleic acid (5,30,48) as well as to displace bonded moieties along their path without specifically interacting with these moieties (5)(6)(7)(8). Thus, unidirectional translocation is a basic activity of helicases and base pair separation might occur as a consequence of the movement.…”

Section: Discussionmentioning

confidence: 99%

“…The mechanisms of these critical processes of the helicase reaction are largely unknown. It is becoming evident that helicases can move unidirectionally along nucleic acid and displace bonded moieties along their path without specifically interacting with these moieties (5)(6)(7)(8). Thus, unidirectional translocation is a basic activity that helicases can perform without requiring interactions with the duplex DNA.…”

mentioning

confidence: 99%

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Jeong

Levin

Patel

2004

Proc. Natl. Acad. Sci. U.S.A.

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Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on singlestranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp͞s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.H elicases are ubiquitous proteins that are involved in various DNA and RNA metabolic processes that require the separation of double-stranded (ds)DNA into single strands, the removal of secondary structures in RNA, or the dissociation of proteins from nucleic acids (1-4). To perform these functions, helicases use the chemical energy from NTP hydrolysis to drive the mechanical processes of translocation and nucleic acid strand separation. In this paper, we study the mechanism of DNA unwinding by bacteriophage T7 helicase that is involved in DNA replication.During replication, the helicase has to unwind a long stretch of DNA, and that requires the helicase to couple strandseparation activity to translocation. The mechanisms of these critical processes of the helicase reaction are largely unknown. It is becoming evident that helicases can move unidirectionally along nucleic acid and displace bonded moieties along their path without specifically interacting with these moieties (5-8). Thus, unidirectional translocation is a basic activity that helicases can perform without requiring interactions with the duplex DNA. Nucleic acid strand separation is a thermodynamically unfavorable process and it is made feasible by the binding of the helicase to the newly unwound strands. Numerous mechanisms of unwinding have been proposed (2-4, 9-13), but additional experimental data are needed to distinguish between these mechanisms. For the monomeric or dimeric helicases such as the Escherichia coli PcrA and Rep helicases (14-16), it has been proposed that unwinding occurs by an active mechani...

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“…Translocation on ssDNA was assessed using an assay, developed by Raney and colleagues (20,21), which measures the helicase catalyzed displacement of streptavidin from a biotinylated 32 P-oligonucleotide. The streptavidin-bound biotinylated DNA substrate 5Ј-[ 32 P]T(bio)GGC-GACGGCAGCGAGGCTTTTTTTTTTTTTTTTTTTT-3Ј (5 nM) and various concentrations of HCV helicase were incubated in reaction buffer (25 mM MOPS, pH 6.5, 3 mM MgCl 2 , 0.1% Tween 20, 37°C) for 30 min.…”

Section: Translocation Assaysmentioning

confidence: 99%

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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DNA Helicases Displace Streptavidin from Biotin-Labeled Oligonucleotides

Cited by 129 publications

References 43 publications

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

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

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

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

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