Reaction conditions in Figure 3 were similar to those in Figure 2A but using 20 nM CMG and 40 nM Mcm10 (where indicated). CMG was mixed with the substrate on ice in the absence of ATP and placed at 30° C for 10’ to allow CMG to load onto the substrates without unwinding. To start the reaction, ATP was added with or without Mcm10 (as indicated). 1’ after starting the reaction, 50 nM unlabeled lagging strand oligo was added as a trap to prevent re-annealing of unwound radiolabeled DNA. Total reaction volumes were 126 μl, and 11 μl aliquots were stopped at the indicated times after addition of ATP and processed as described for the assays of Figure 2A.