Figure 1.

The characteristic unwinding dynamics of E1. (A) Two kinds of E1 constructs were used. One is E1 (residue 128–605), which consists of a DBD and an HD. The other is E1HD (residue 308–605), which contains only an HD. (B) Schematics of the magnetic tweezers assay to monitor the unwinding of E1. While HP is pulled by a pair of magnets at a force lower than F_unzip, E1 monomers and ATP-Mg²⁺ are flushed into the chamber. E1 assembles into an active hexameric form and then begins to unwind HP. The resultant extension increase (Δz) is monitored and converted into the number of unwound base pairs. (C) A representative unwinding trace of E1 on HP50bp at 5 pN. E1 could fully unwind HP50bp. After reaching the maximum, HP was gradually rewound because of E1 translocating past the hairpin apex. (D) E1 showed diverse dynamics from monotonic single unwinding (upper two panel) to repetitive unwinding (lower two panel). Between two unwinding motions, repetitive motion is more dominant, comprising 65%. In repetitive motion, E1 moved back through either abrupt slippage or gradual rewinding. (E) To assess the unwinding processivity of E1, we performed the experiment with a longer HP (HP1kbp). The number of base pairs unwound by E1 within 500 s was broadly distributed from several tens (upper two panels) to more than 100 bp (lower panel) at 5 pN. However, the maximum number of unwound base pairs was, in general, below 180 bp.