(A) Titration of Mcm10 in a CMG unwinding assay. Reactions contained 25 nM CMG with either: no Mcm10 (lanes 2–4), 25 nM Mcm10 (1:1, lanes 5–7), 50 nM Mcm10 (2:1, lanes 8–10) or 100 nM Mcm10 (4:1, lanes 11–13). See Materials and methods for details. Also see Figure 2—figure supplements 1–2. (B) Quantification of the data from (A). Values are the average of three independent experiments and the error bars show the standard deviation. (C) The same data displayed as a dose-response curve of unwinding as a function of the Mcm10:CMG ratio in the reaction at the three different time points. (D) Isolation of CMG-Mcm10 complex using CMG-Flag and anti-Flag beads. CMG and a 3-fold molar excess of Mcm10 were mixed, attached to beads, then washed twice and eluted with Flag peptide. The PAGE gel shows elution from the anti-Flag beads that were loaded with either CMG-flag alone (lane 1), Mcm10 alone (lane 2), or the CMG-flag +Mcm10 mixture (lane 3). The protein bands are identified to the right. A densitometric scan of the bands in the gel of the CMG-Mcm10 complex indicates a molar ratio of 1.8 Mcm10 per Mcm2-7 complex; the Cdc45 is typically in excess due to the Flag tag (i.e. any dissociated Cdc45 will bind the beads). The GINS subunits ran off the gel (see Figure 2—figure supplements 3–4 for GINS stoichiometry). Also see Figure 2—figure supplement 5 for helicase activity of purified CMG-Mcm10 complex.