For the assays in Figure 5, the forked DNA was formed by annealing 50 duplex LAG dual biotin and 5’-32P-50duplex LEAD (see Table 1). The biotinylated dT nucleotides are 13 and 20 bases from the forked junction. For the assays in Figure 5—figure supplement 1, the forked DNA was formed by annealing 50 duplex LEAD2 dual biotin and 5’-32P-50duplex LAG2 (see Table 1). The biotinylated dT nucleotides are 12 and 19 bases from the forked junction. Oligos were annealed as described above. Reaction conditions were similar to those in Figure 3 except that the final reaction volume was 45 μl and 4 μg/ml streptavidin was added (where indicated) during the 10’ CMG pre-incubation. CMG was at 25 nM and Mcm10 or MTC was at 50 nM (final concentrations) when present. In these assays, the trap oligo was 50 nM unlabeled 50duplex LEAD oligo. 12 μl aliquots were removed at the indicated times after addition of ATP, terminated with EDTA/SDS stop buffer, flash frozen and processed as above. For the assays in Figure 6, the radiolabel was on the biotinylated strand and, where indicated, free biotin was added at the start of the reaction (after streptavidin pre-incubation) to a final concentration of 1.5 μM to prevent re-binding of any streptavidin displaced by CMG/Mcm10 during the unwinding reaction.