(A) Scheme of the reaction. CMG was pre-incubated with the substrates for 10’ before addition of ATP ± a 2-fold excess of Mcm10 over CMG. (B) Native PAGE analysis of CMG unwinding in the absence (lanes 2–10) or presence (lanes 11–19) of Mcm10 using either a 160 bp duplex fork (panel B) or a 50 bp duplex fork (panel C) as indicated by the schematics to the left of the gels. (D) Quantification of the data in the gels. (E) The first 5’ of reactions with Mcm10 are shown to better illustrate the effect of Mcm10 on unwinding by CMG. The values are averages of three independent experiments and the error bars show the standard deviation. Also see Figure 3—figure supplement 1.