Figure 5.

NS3h‐mediated repetitive‐looping. A: Cy3 intensity increases as NS3h approaches. Two populations were observed in single‐molecule time traces in smFRET experiments. Subpopulation 1: the fluorescence intensity of Cy3 fluorophore shows a gradual increase followed by a quick drop repeatedly which is caused by the PIFE effect. smFRET time traces were recorded in the presence of 2 mM ATP and 2 nM NS3h. Donor (Cy3) intensity is shown in green, acceptor (Cy5) intensity in red, and FRET efficiency in blue. B: FRET values show the distance changes between the donor fluorophore at the 5′‐tail and the acceptor fluorophore at the partial duplex junction. Subpopulation 2 shows repetitive cycling in single‐molecule time traces, in addition. C: Repetitive translocation model. (II) NS3h binds at the 5′ partial duplex junction and starts to translocate along the ssDNA, d(T)65, until it reaches the 5′end (Cy3). Once NS3h reaches the end, it snaps back to the duplex junction and re‐initiates translocation. D: Reeling‐in model. (I) NS3h binds to the partial duplex junction. (II) Translocation starts in the 3′ to 5′ direction while NS3h stays at the duplex junction. (III) The size of the ssDNA loop formed increases while NS3h continues to translocate to the 5′‐tail. (IV) NS3h reaches the end of the nucleic acid and dissociates. (I)–(IV) are repeated for numerous cycles. E: Histogram of time intervals of the repetitions (Δt) shown in (A) and fitted using a Gaussian function (black curve; mean time interval: 2.15 ± 0.03 s). F: Histogram of time intervals of the repetitions (Δt) shown in (B) and fitted using a Gaussian function (black curve; mean time interval: 1.70 ± 0.05 s).