Coupling Translocation with Nucleic Acid Unwinding by NS3 Helicase

Abstract

NS3 helicase from hepatitis C virus (HCV) uses the free energy from ATP hydrolysis to translocate along single stranded DNA (ssDNA) or RNA, unwind double-stranded (ds) duplex, or displace proteins along the track. In this work, we present a semi-quantitative model for translocation and unwinding activities of monomeric NS3 helicase. The model is based on structural, biochemical and single molecule measurements of the helicase. First, we have assumed that NS3 helicase walks unidirectionally by alternately moving its two translocase domains forward one nucleotide (nt) at a time, as that being suggested for a similar helicase PcrA. Second, in our model NS3 helicase displays diffusive character upon ATP binding (before helicase dissociation), due to weak affinity of the helicase to the ssDNA. The helicase diffusion leads to occasional backward steps and increases displacement fluctuations that are supposedly detectable from single molecule experiments. Further, our model suggests that NS3 helicase interacts with the duplex at the ss-ds junction such that (i) the helicase can actively unwind the duplex by reducing free energy of base pairing /stacking at the duplex end; (ii) the helicase can be stabilized at the junction by its favorable association with the duplex backbone. By fitting with NS3 unwinding data from single molecule optical-tweezer measurements, our model explains sequence dependences of the unwinding velocity, efficiency, and helicase dissociation rate. In particular, based on the experimental data, we have quantified how active the NS3 helicase is in its unwinding, and estimated how fast the helicase diffuses in its ATP bound state. The generic features of coupling ssDNA/RNA translocation with duplex unwinding presented in our NS3 helicase model may apply as well to other similar systems.