Nucleic Acids Research, 2001, Vol. 29, No. 2 565-572
© 2001 Oxford University Press
Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA and 1Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16803, USA
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.
* To whom correspondence should be addressed. Tel: +1 501 686 5244; Fax: +1 501 686 8169; Email: raneykevind{at}exchange.uams.edu
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