Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase.
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.
Myong
Sua
S
Physics Department, University of Illinois, 1110 West Green Street, Urbana, IL 61801, USA. smyong@uiuc.edu
Bruno
Michael M
MM
Pyle
Anna M
AM
Ha
Taekjip
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D020029:Base Pairing; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D016174:Hepacivirus / Q000201:enzymology*; D008954:Models, Biological; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases