DNA Unwinding Mechanism of the Helicase From Hepatitis C Virus
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Hepatitis C virus (HCV) ssRNA genome includes non-structural protein 3, a superfamily 2 helicase (NS3h). NS3h uses the energy of ATP hydrolysis to separate the strands of dsDNA or dsRNA. This process is essential for viral replication and represents a potential target for antiviral therapy. In this work, thermodynamic, kinetic, and structure-function approaches were employed to elucidate the DNA unwinding mechanism of HCV helicase.
NS3h interactions with DNA were studied by fluorimetric titration and electron microscopy. NS3h was shown to bind ssDNA with a minimal binding site of 8 bases, an occlusion site of 6 to 7 bases, and an intrinsic Kd of 2 nM, while the affinity of NS3h-ATP complex for ssDNA is 260 fold weaker. Thus, the ATPase cycle controls the NS3h interactions with DNA.
The NS3h unwinding kinetics was studied under both multiple and single turnover conditions. The increase in the rate of DNA unwinding with increasing NS3h protein concentration under multiple turnover conditions together with mutant NS3h inhibition of unwinding provides evidence that many NS3h proteins are involved in unwinding of a single DNA substrate.
The efficiency of the unwinding under single turnover conditions increased with the length of ssDNA tail of the unwinding substrate, but decreased with the length of the duplex DNA. The number of NS3h molecules bound to ssDNA tail of a ss/dsDNA unwinding substrate is proportional to the length of the tail. These results indicate that multiple helicase molecules are required for efficient unwinding, while global data fitting revealed that the unwinding processivity of a single helicase molecule is low (89% per base) and multiple helicase molecules are required to prevent the DNA strands from reannealing behind the helicase complex.
Finally, I propose a mechanism for translocation and unwinding of HCV helicase, which is consistent with the Brownian motor model: Binding to the ss/dsDNA substrate causes the helicase to move in the 3' to 5' direction, ATP binding makes it partially release the DNA. Upon ATP hydrolysis, the helicase rebinds the DNA while making a step forward. This general mechanism should be applicable to other helicases.
Document number: osu1017851412
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1017851412
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