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Protein Sci. 2017 Jul;26(7):1391-1403. doi: 10.1002/pro.3136. Epub 2017 Mar 6.

Single-molecule imaging reveals the translocation and DNA looping dynamics of hepatitis C virus NS3 helicase.

Author information

1
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland.
2
Department of Physics Education, Kongju National University, Kongju, 32588, Republic of Korea.
3
Center for Free-Electron Laser Science, DESY, 22607, Hamburg, Germany.
4
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York.
5
Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland.
6
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland.
7
Howard Hughes Medical Institute, Baltimore, Maryland.

Abstract

Non-structural protein 3 (NS3) is an essential enzyme and a therapeutic target of hepatitis C virus (HCV). Compared to NS3-catalyzed nucleic acids unwinding, its translation on single stranded nucleic acids have received relatively little attention. To investigate the NS3h translocation with single-stranded nucleic acids substrates directly, we have applied a hybrid platform of single-molecule fluorescence detection combined with optical trapping. With the aid of mechanical manipulation and fluorescence localization, we probed the translocase activity of NS3h on laterally stretched, kilobase-size single-stranded DNA and RNA. We observed that the translocation rate of NS3h on ssDNA at a rate of 24.4 nucleotides per second, and NS3h translocates about three time faster on ssRNA, 74 nucleotides per second. The translocation speed was minimally affected by the applied force. A subpopulation of NS3h underwent a novel translocation mode on ssDNA where the stretched DNA shortened gradually and then recovers its original length abruptly before repeating the cycle repetitively. The speed of this mode of translocation was reduced with increasing force. With corroborating data from single-molecule fluorescence resonance energy transfer (smFRET) experiments, we proposed that NS3h can cause repetitive looping of DNA. The smFRET dwell time analysis showed similar translocation time between sole translocation mode versus repetitive looping mode, suggesting that the motor domain exhibits indistinguishable enzymatic activities between the two translocation modes. We propose a potential secondary nucleic acids binding site at NS3h which might function as an anchor point for translocation-coupled looping.

KEYWORDS:

NS3 helicase; florescence localization; optical trapping; repetitive looping; single-molecule detection; translocation

PMID:
28176403
PMCID:
PMC5477530
DOI:
10.1002/pro.3136
[Indexed for MEDLINE]
Free PMC Article

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