英文原文链接：
http://www.sciencedaily.com/releases/2007/07/070726142007.htm
原始出处:
Science27 July 2007:
Vol. 317. no. 5837, pp. 513 - 516
DOI: 10.1126/science.1144130
Spring-Loaded Mechanism of DNA Unwinding by Hepatitis C Virus NS3 Helicase
Sua Myong,1,2* Michael M. Bruno,3,4 Anna M. Pyle,3,4 Taekjip Ha1,2,4
NS3, an essential helicase for replication of hepatitis C virus, is a model enzyme for investigating helicase function. Using single-molecule fluorescence analysis, we showed that NS3 unwinds DNA in discrete steps of about three base pairs (bp). Dwell time analysis indicated that about three hidden steps are required before a 3-bp step is taken. Taking into account the available structural data, we propose a spring-loaded mechanism in which several steps of one nucleotide per adenosine triphosphate molecule accumulate tension on the protein-DNA complex, which is relieved periodically via a burst of 3-bp unwinding. NS3 appears to shelter the displaced strand during unwinding, and, upon encountering a barrier or after unwinding >18 bp, it snaps or slips backward rapidly and repeats unwinding many times in succession. Such repetitive unwinding behavior over a short stretch of duplex may help to keep secondary structures resolved during viral genome replication.
1 Physics Department, University of Illinois, 1110 West Green Street, Urbana, IL 61801, USA.
2 Institute for Genomic Biology, University of Illinois, 1206 West Gregory Drive, Urbana, IL 61801, USA.
3 Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, Room 334A, Bass Building, New Haven, CT 06511, USA.
4 Howard Hughes Medical Institute.
* To whom correspondence should be addressed. E-mail:smyong@uiuc.edu
Fig. 1. NS3 unwinds DNA in 3-bp steps. (A) PD1, a DNA with 18 double strands (ds) and 20 single strands (ss), was labeled with donor (Cy3) and acceptor (Cy5) at the ds-ss junction and was tethered to a polyethylene glycol surface by 3' biotin. (B) Cy3 (green) and Cy5 (red) intensities monitored during unwinding of a single PD1 molecule (raw time traces in light color, three-point averaged traces in dark color). (C) Calculated FRET efficiency versus time for the molecule shown in (B). (D and E) Two more examples of FRET traces of PD1. (F) PD2 is thesameconstruct as in (A) but prepared with dyes in the middle of duplex. (G to J) Plots analogous to those in (B) to (E) for PD2.