Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro

Alan J. Tackett, Yingfeng Chen, Craig Eugene Cameron, Kevin D. Raney

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

NS3 (nonstructural protein 3) from the hepatitis C virus is a 3′ → 5′ helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3. When the enzyme was in excess of the substrate, NS3 (500 nM) unwound >80% of a DNA substrate containing a 15-nucleotide overhang and a 30-bp duplex (45:30-mer; 1 nM). This result indicated that the active form of NS3 that was bound to the DNA prior to initiation of the reaction was capable of processive DNA unwinding. Unwinding with varying ratios of NS3 to 45:30-mer allowed us to investigate the active form of NS3 during the first unwinding cycle. When the substrate concentration slightly exceeded that of the enzyme, little or no unwinding was observed, indicating that if a monomeric form of the protein is active, then it exhibits very low processivity. Binding of NS3 to the 45:30-mer was measured by electrophoretic mobility shift assays, resulting in KD = 2.7 ± 0.4 nM. Binding to individual regions of the substrate was investigated by measuring the KD for a 15-mer oligonucleotide as well as a 30-mer duplex. NS3 bound tightly to the 15-mer (KD = 1.3 ± 0.2 nM) and, surprisingly, fairly tightly to the double-stranded 30-mer (KD = 11.3 ± 1.3 nM). However, NS3 was not able to rapidly unwind a blunt-end duplex. Thus, under conditions of optimal unwinding, the 45:30-mer is initially saturated with the enzyme, including the duplex region. The unwinding data are discussed in terms of a model whereby multiple molecules of NS3 bound to the single-stranded DNA portion of the substrate are required for optimal unwinding.

Original languageEnglish (US)
Pages (from-to)10797-10806
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number11
DOIs
StatePublished - Mar 18 2005

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Oligonucleotides
Molecules
DNA
Proteins
Substrates
In Vitro Techniques
Enzymes
Assays
Electrophoretic mobility
Single-Stranded DNA
Electrophoretic Mobility Shift Assay
Hepacivirus
Viruses
Carrier Proteins
Nucleotides

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro",
abstract = "NS3 (nonstructural protein 3) from the hepatitis C virus is a 3′ → 5′ helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3. When the enzyme was in excess of the substrate, NS3 (500 nM) unwound >80{\%} of a DNA substrate containing a 15-nucleotide overhang and a 30-bp duplex (45:30-mer; 1 nM). This result indicated that the active form of NS3 that was bound to the DNA prior to initiation of the reaction was capable of processive DNA unwinding. Unwinding with varying ratios of NS3 to 45:30-mer allowed us to investigate the active form of NS3 during the first unwinding cycle. When the substrate concentration slightly exceeded that of the enzyme, little or no unwinding was observed, indicating that if a monomeric form of the protein is active, then it exhibits very low processivity. Binding of NS3 to the 45:30-mer was measured by electrophoretic mobility shift assays, resulting in KD = 2.7 ± 0.4 nM. Binding to individual regions of the substrate was investigated by measuring the KD for a 15-mer oligonucleotide as well as a 30-mer duplex. NS3 bound tightly to the 15-mer (KD = 1.3 ± 0.2 nM) and, surprisingly, fairly tightly to the double-stranded 30-mer (KD = 11.3 ± 1.3 nM). However, NS3 was not able to rapidly unwind a blunt-end duplex. Thus, under conditions of optimal unwinding, the 45:30-mer is initially saturated with the enzyme, including the duplex region. The unwinding data are discussed in terms of a model whereby multiple molecules of NS3 bound to the single-stranded DNA portion of the substrate are required for optimal unwinding.",
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Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro. / Tackett, Alan J.; Chen, Yingfeng; Cameron, Craig Eugene; Raney, Kevin D.

In: Journal of Biological Chemistry, Vol. 280, No. 11, 18.03.2005, p. 10797-10806.

Research output: Contribution to journalArticle

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