In Figure 2A we titrated Mcm10 into a helicase assay using a fixed amount of CMG and a synthetic forked DNA with a 50 bp duplex and 40-nt dT ssDNA tails. Stimulation by Mcm10 is remarkable, with over 30-fold enhancement of CMG unwinding at 2’ in the presence of only one molecule of Mcm10 per CMG (compare lane 5 to lane 2 in Figure 2A). Reactions containing Mcm10 are complete by the 5 min time point, while the CMG reaction without Mcm10 continues slow unwinding during the 10 min time course (Figure 2B). The results at different Mcm10 concentrations indicate that maximal stimulation of CMG is observed with two subunits of Mcm10 for each CMG complex and further addition of Mcm10 beyond this amount has little effect. (Figure 2C). Control reactions with Mcm10 alone, lacking CMG, show no unwinding activity (Figure 2—figure supplement 1). Reactions with Mcm10 sometimes give a supershift of some of the substrate (e.g. lanes 5, 8, 11, 12 in Figure 2A) which we interpret as a gel shift of Mcm10 that remains bound to DNA, as Mcm10 is a known DNA binding protein (Du et al., 2012; Robertson et al., 2008; Warren et al., 2008). To determine if other DNA binding proteins such as RPA or E. coli SSB stimulate CMG, we performed experiments in which CMG unwinding was initiated and then either Mcm10, RPA or SSB was added. The results show that the stimulation of CMG is specific to Mcm10 and that RPA and SSB do not substitute for Mcm10 (Figure 2—figure supplement 2).