Fuel Specificity of the Hepatitis C Virus NS3 Helicase

被引:20
作者
Belon, Craig A. [1 ]
Frick, David N. [1 ]
机构
[1] New York Med Coll, Dept Biochem & Mol Biol, Valhalla, NY 10595 USA
基金
美国国家卫生研究院;
关键词
viral replication; motor protein; ATPase; RNA; DNA; RNA HELICASE; STRAND SEPARATION; CRYSTAL-STRUCTURE; DUPLEX RNA; PROTEIN; DNA; MECHANISM; DOMAIN; TRANSLOCATION; REPLICATION;
D O I
10.1016/j.jmb.2009.03.059
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of all oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in Solution, Such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTR 3'-Deoxy-NTPs generally Support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of all intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP Supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson-Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2' and 3' carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn2+ supporting about 10 times faster unwinding than Mg2+. Unlike Mg2+, Mn2+ does not Support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction. (C) 2009 Elsevier Ltd. All rights reserved.
引用
收藏
页码:851 / 864
页数:14
相关论文
共 49 条
[1]   Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase [J].
Ali, JA ;
Lohman, TM .
SCIENCE, 1997, 275 (5298) :377-380
[2]   Monitoring helicase activity with molecular beacons [J].
Belon, Craig A. ;
Frick, David N. .
BIOTECHNIQUES, 2008, 45 (04) :433-+
[3]   Direct fluorometric measurement of hepatitis C virus helicase activity [J].
Boguszewska-Chachulska, AM ;
Krawczyk, M ;
Stankiewicz, A ;
Gozdek, A ;
Haenni, AL ;
Strokovskaya, L .
FEBS LETTERS, 2004, 567 (2-3) :253-258
[4]   Inhibition of hepatitis C virus RNA replication by 2′-modified nucleoside analogs [J].
Carroll, SS ;
Tomassini, JE ;
Bosserman, M ;
Getty, K ;
Stahlhut, MW ;
Eldrup, AB ;
Bhat, B ;
Hall, D ;
Simcoe, AL ;
LaFemina, R ;
Rutkowski, CA ;
Wolanski, B ;
Yang, ZC ;
Migliaccio, G ;
De Francesco, R ;
Kuo, LC ;
MacCoss, M ;
Olsen, DB .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (14) :11979-11984
[5]   Crystal structure of RNA helicase from genotype 1b hepatitis C virus - A feasible mechanism of unwinding duplex RNA [J].
Cho, HS ;
Ha, NC ;
Kang, LW ;
Chung, KM ;
Back, SH ;
Jang, SK ;
Oh, BH .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (24) :15045-15052
[6]   Helicase mechanisms and the coupling of helicases within macromolecular machines Part 1: Structures and properties of isolated helicases [J].
Delagoutte, E ;
von Hippel, PH .
QUARTERLY REVIEWS OF BIOPHYSICS, 2002, 35 (04) :431-478
[7]   RNA translocation and unwinding mechanism of HCVNS3 helicase and its coordination by ATP [J].
Dumont, S ;
Cheng, W ;
Serebrov, V ;
Beran, RK ;
Tinoco, I ;
Pyle, AM ;
Bustamante, C .
NATURE, 2006, 439 (7072) :105-108
[8]  
Flicker RDJ., 2007, ADV DIS, V3, P1
[9]   Understanding helicases as a means of virus control [J].
Frick, D. N. ;
Lam, A. M. I. .
CURRENT PHARMACEUTICAL DESIGN, 2006, 12 (11) :1315-1338
[10]  
Frick DN, 2007, CURR ISSUES MOL BIOL, V9, P1