Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro

Tani, Hidenori; Fujita, Osamu; Furuta, Atsushi; Matsuda, Yoshiki; Miyata, Ryo; Akimitsu, Nobuyoshi; Tanaka, Junichi; Tsuneda, Satoshi; Sekiguchi, Yuji; Noda, Naohiro

doi:10.1016/j.bbrc.2010.01.100

Cited by 32 publications

(42 citation statements)

References 25 publications

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“…Screening is conducted using a FRET-based fluorescence helicase assay [17] (see Note 12). Here, we describe an evaluation of the effects of test compounds on fluorescence intensity that can also be adapted to identify false-positive and false-negative hits interfering with the FRET-based assay.…”

Section: Methodsmentioning

confidence: 99%

“…Because the assay does not require electrophoretic separation of the strands unwound by the helicase, it can be readily adapted for high-throughput screening or for measurement of real-time kinetics [14][15][16]. We previously applied the assay to the monitoring of NS3 helicase activity, using a dsRNA substrate [17]. To prepare the dsRNA substrate, a 5′ fluorophore-labeled oligoribonucleotide is hybridized to a 3′ quencher-labeled oligoribonucleotide, yielding a dsRNA substrate with a 3′ single-stranded overhang.…”

Section: Introductionmentioning

confidence: 99%

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A Fluorescence-Based Screening Assay for Identification of Hepatitis C Virus NS3 Helicase Inhibitors and Characterization of Their Inhibitory Mechanism

Furuta

Salam

Tani

et al. 2014

Methods in Molecular Biology

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Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug-drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Fluorescence-Based Screening Assay for Identification of Hepatitis C Virus NS3 Helicase Inhibitors and Characterization of Their Inhibitory Mechanism

Furuta

Salam

Tani

et al. 2014

Methods in Molecular Biology

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“…Helicase Assays-The helicase activity was based on FRET, by using a double-stranded substrate (Table 4) with a fluorophore (6FAM) on one strand and a quencher (BHQ1) on the other, and by monitoring the fluorence emitted when the two strands are separated by the helicase action (33). The assay was performed in 20 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 50 mM KCl, 8 mM DTT, 0.1 mg/ml BSA, and 5% glycerol (v/v) with 10 or 100 nM dsDNA substrate (F1:F2 and B1:B2), 3 mM ATP, and 125 nM capture strand (to prevent reannealing) in 30 l of reaction volume.…”

Section: Methodsmentioning

confidence: 99%

The Human RecQ4 Helicase Contains a Functional RecQ C-terminal Region (RQC) That Is Essential for Activity

Mojumdar

March

Marino

et al. 2017

Journal of Biological Chemistry

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Edited by F. Peter GuengerichRecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasmaatomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix ␤-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.

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“…When the dsRNA substrate is unwound by the helicase, the dye emits fluorescence upon its release from the quencher (58).…”

Section: Proteomics Identifies Novel Binding Partners Of Ezrinmentioning

confidence: 99%

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

Çelik

Sajwan

Selvanathan

et al. 2015

Molecular and Cellular Biology

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Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5= untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane. Ezrin is a prototype member of the ERM (ezrin-radixin-moesin) family of proteins that functions as a scaffold between the plasma membrane and the underlying cortical actin cytoskeleton (1, 2). Ezrin regulates cytoskeletal dynamics in response to both internal and external stimuli through its intracellular localization and protein binding activities; thus, it plays an important role in the maintenance of cell shape, cell polarity, adhesion, and movement (3). All members of the ERM family are characterized by the presence of a shared FERM domain at the amino terminus, which can bind to transmembrane proteins, including CD43, CD44, CD95, ICAMs, syndecan 2, EBP50/NHERF1, and E3KARP/NHERF2. The carboxy termini of ERM proteins contain an F-actin binding domain, which both regulates intramolecular interactions with amino-terminal FERM domains and promotes F-actin organization. The pleiotropic functions of ezrin in a wide range of cellular processes can be explained through its association with numerous proteins with diverse functions (4). Several lines of evidence have indicated that ezrin can oscillate between various "open/active" and "closed/dormant" states, which are regulated by self-association of N-terminal and Cterminal regions. Head-to-tail folding of the molecule likely masks the respective protein binding sites and leads to the localization of ezrin in the cytoplasm in its monomeric form. The conformational switch to an open state requires direct interaction with the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and phosphorylation of a conserved threonine (T567) located in the ...

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Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro

Cited by 32 publications

References 25 publications

A Fluorescence-Based Screening Assay for Identification of Hepatitis C Virus NS3 Helicase Inhibitors and Characterization of Their Inhibitory Mechanism

A Fluorescence-Based Screening Assay for Identification of Hepatitis C Virus NS3 Helicase Inhibitors and Characterization of Their Inhibitory Mechanism

The Human RecQ4 Helicase Contains a Functional RecQ C-terminal Region (RQC) That Is Essential for Activity

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

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