10.7554/eLife.29118.010Figure 3—figure supplement 1.Delayed replication of longer duplex allows estimation of unwinding rate.A helicase assay identical to those in Figure 3 was performed except that both the 50 bp duplex and 160 bp duplex for substrates were added to the reaction at 0.5 nM each substrate and pre-incubated together with 20 nM CMG in the absence of ATP as shown in the scheme above the gel. The reaction was started by the addition of ATP (1 nM) and Mcm10 (40 nM) and unlabeled traps for each substrate (25 nM) were added 1’ later to prevent re-annealing of unwound DNA. The total reaction volume was 91 μl and at the indicated times, 11 μl aliquots were removed, stopped with buffer containing 20 mM EDTA and 0.1% SDS (final concentrations), and flash frozen in liquid nitrogen. The positions of the two substrates are indicated in lanes 1–3 of the gel showing that they migrate at distinguishable positions. Unwinding of each substrate was calculated as [unwound/(unwound + unreacted substrate)] for each substrate separately and the values are shown in the graph below the gel.