Leading strand replication experiments in Figure 4 used a singly primed 2.8 kb forked linear DNA substrate that was previously described (Georgescu et al., 2014). The duplex portion of the DNA substrate is linearized pUC19 DNA to which a synthetic fork junction has been ligated to one end of the duplex. The fork is primed for leading strand DNA replication with 5’-32 P-C2 oligo (Table 1). Reactions were 25 μL and contained 30 nM CMG, 10 nM Pol ε, 5 nM RFC, 25 nM PCNA, 600 nM RPA and 1.25 nM linear forked template (final concentrations) in a buffer consisting of 25 mM Tris Acetate pH 7.5, 5% glycerol, 40 μg/ml BSA, 3 mM DTT, 2 mM TCEP, 10 mM magnesium acetate, 50 mM K glutamate, 0.1 mM EDTA, 5 mM ATP, and 120 μM of each dNTP. Replication assays were performed by first incubating CMG (and the indicated amount of Mcm10 and/or MTC, where indicated) with linear forked template for 5’ at 30° C, followed by addition of RFC, PCNA, and Pol ε for 4’ in the presence of dATP and dCTP to support clamp loading and polymerase binding while preventing 3’−5’ exonuclease activity on the primer. Reactions were started by addition of ATP, RPA, and the withheld nucleotides (dGTP and dTTP). The reactions proceeded for the indicated amount of time at 30°C and were stopped with an equal volume of 2X stop solution (40 mM EDTA and 1% SDS). Reaction products were analyzed on 1.3% alkaline agarose gels at 35 V for 17 hr, backed with DE81 paper, and dried by compression. Gels were exposed to a phosphorimager screen and imaged with a Typhoon FLA 9500 (GE Healthcare).