title: Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex.
abstract: Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA-DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA-DNA substrate. Binding of NS3 to the PNA-DNA substrate was similar to the DNA-DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA-RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino-DNA and phosphorothioate-DNA substrates were utilized as efficiently by NS3 as DNA-DNA substrates. These results indicate that the PNA-DNA and PNA-RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Tackett
authors.forename: L
authors.initials: L
authors.lastname: Wei
authors.forename: C E
authors.initials: CE
authors.lastname: Cameron
authors.forename: K D
authors.initials: KD
authors.lastname: Raney
mesh_terms: D002384:Catalysis; D015226:Dinucleoside Phosphates / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology*; D006358:Hot Temperature; D009025:Morpholines / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000378:metabolism*; D009696:Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D015226:Dinucleoside Phosphates; D009025:Morpholines; D009692:Nucleic Acid Heteroduplexes; D009696:Nucleic Acids; D009838:Oligodeoxyribonucleotides; D020365:RNA Helicases; D013873:Thionucleotides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases