For the experiments in Figure 5 using a stalled replisome, the substrate was made by annealing Blocked Fork LEAD, Blocked Fork LAG and 5’-32P-Blocked Fork Primer (Table 1). The lagging strand oligo has two biotinylated dT nucleotides that are 13 and 20 bases from the forked junction. To form the lagging strand block, streptavidin (75 nM) was added to the substrate (1.25 nM) and incubated at 30°C for 5’ in buffer consisting of 20 mM Tris Acetate, 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 40 μg/mL BSA and 10 mM MgSO4 (all amounts are the final concentration in the complete reaction). Next, 30 nM CMG was added along with 0.5 mM ATP for 3’ to allow CMG to translocate to the block and then replication was initiated by adding 5 nM RFC, 20 nM PCNA, 20 nM Pol ε, 5 mM ATP, and 30 nM MTC (when present) along with adding 115 μM of each dNTP. After a further 2’ incubation, Mcm10 was either added at 60 nM or omitted. Aliquots of each reaction were collected 1, 3, and 10 min later and quenched with an equal volume of Stop Buffer containing 78% formamide, 8 mM EDTA, and 1% SDS. Samples were boiled and then analyzed by PAGE in a 10% Urea gel. Gels were washed in distilled water, mounted on Whatman 3 MM paper, wrapped in plastic and exposed to a storage phosphor screen that was scanned on a Typhoon 9400 laser imager (GE Healthcare).