title: Dynamic Interconversions of HCV Helicase Binding Modes on the Nucleic Acid Substrate.
abstract: The dynamics involved in the interaction between hepatitis C virus nonstructural protein 3 (NS3) C-terminal helicase and its nucleic acid substrate have been the subject of interest for some time given the key role of this enzyme in viral replication. Here, we employed fluorescence-based techniques and focused on events that precede the unwinding process. Both ensemble Förster resonance energy transfer (FRET) and ensemble protein induced fluorescence enhancement (PIFE) assays show binding on the 3' single-stranded overhang of model DNA substrates (>5 nucleotides) with no preference for the single-stranded/double-stranded (ss/ds) junction. Single-molecule PIFE experiments revealed three enhancement levels that correspond to three discrete binding sites at adjacent bases. The enzyme is able to transition between binding sites in both directions without dissociating from the nucleic acid. In contrast, the NS3 mutant W501A, which is unable to engage in stacking interactions with the DNA, is severely compromised in this switching activity. Altogether our data are consistent with a model for NS3 dynamics that favors ATP-independent random binding and sliding by one and two nucleotides along the overhang of the loading strand.
authors.affiliation: Department of Biochemistry, McGill University , Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ablenas
authors.affiliation: Department of Microbiology and Immunology, McGill University , Montreal, Quebec H3A 2B4, Canada.
authors.forename: Hsiao-Wei
authors.initials: HW
authors.lastname: Liu
authors.affiliation: Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Nikoloz
authors.initials: N
authors.lastname: Shkriabai
authors.affiliation: Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Mamuka
authors.initials: M
authors.lastname: Kvaratskhelia
authors.affiliation: Department of Chemistry and the Centre for Self-Assembled Chemical Structures (CSACS/CRMAA), McGill University , Montreal, Quebec H3A 2K6, Canada.
authors.forename: Gonzalo
authors.identifier: 0000-0003-0064-1345
authors.initials: G
authors.lastname: Cosa
authors.affiliation: Department of Microbiology and Immunology, McGill University , Montreal, Quebec H3A 2B4, Canada.|Department of Medical Microbiology and Immunology, University of Alberta , Edmonton, Alberta T6G 2E1, Canada.|Department of Medicine, Division of Experimental Medicine, McGill University , Montreal, Quebec H3A 1A3, Canada.
authors.forename: Matthias
authors.identifier: 0000-0001-6213-5806
authors.initials: M
authors.lastname: Götte
mesh_terms: D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism; D005838:Genotype; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D009696:Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D004265:DNA Helicases; D009696:Nucleic Acids; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
keywords: DNA helicase; RNA helicase; fluorescence resonance energy transfer (FRET); hepatitis C virus (HCV); protein induced fluorescence enhancement (PIFE)