Figure 3.

Kymographs of BfAddAB unwinding of YOYO-1-stained lambda DNA at 2 mM Mg²⁺-ATP. Adjacent to each kymograph is a plot of DNA molecule length (kb) with time (seconds) smoothed using a five-point moving average. The BfAddAB enzyme rates were determined by linear least squares fitting of the slope. To calculate the processivity of each individual event, the DNA molecule length was determined before and after unwinding by BfAddAB. DNA in these experiments had a staining ratio of 1:20 dye:bp. (a) A truncated DNA molecule whose BfAddAB-mediated unwinding commenced at 80 s and proceeded to completion. (b) A DNA molecule whose unwinding stalled presumably due to dissociation of the BfAddAB. (c) DNA unwinding was stopped prematurely because of tether instabilities which results in loss of the active BfAddAB–DNA complex from the field of view. Note that at ∼120 s the right-hand DNA end became stuck to the surface and broke off. (d) Control image. The DNA sample was incubated with BfAddAB but without Mg²⁺-ATP. No unwinding was ever observed, only instantaneous truncations such as that at ∼280 s due to photodamage.