As shown in the schematic at the top, CMG (20 nM) was pre-incubated with the 160-mer duplex substrate from Figure 3B (0.5 nM) for 10’ followed by addition of ATP (1 mM) to start the CMG unwinding reaction. 1’ after adding ATP, unlabeled trap oligo (20 nM) was added to the reaction to prevent re-annealing of any unwound strands. 10’ after addition of ATP, either Mcm10 (red), yeast RPA (blue), or E. coli SSB (green) (‘chase’ protein, 80 nM) was added to the reaction to determine whether it stimulates CMG unwinding. 30’ prior to addition of the chase protein, an aliquot of each reaction was withdrawn to determine the extent of CMG unwinding prior to addition of the chase protein. Time points were also taken 15’, 30’, 1’, 2’ and 5’ after addition of the chase protein, analyzed by gel electrophoresis and quantified as previously described.