Reactions in Figure 2A contained 25 nM CMG and either 0 nM, 25 nM, 50 nM or 100 nM of Mcm10 (as indicated) with 0.5 nM DNA substrate and 1 mM ATP in 40 μl final volume of buffer A (20 mM Tris Acetate pH 7.6, 5 mM DTT, 0.1 mM EDTA, 10 mM MgSO4, 30 mM KCl, 40 μg/ml BSA). Reactions were mixed on ice and started by placing in a water bath at 30° C. 1’ after starting the reaction, 25 nM unlabeled 50duplex LAG oligo was added as a trap to prevent re-annealing of unwound radiolabeled DNA. At the indicated times, 12 μl aliquots were removed, stopped with buffer containing 20 mM EDTA and 0.1% SDS (final concentrations), and flash frozen in liquid nitrogen. Frozen reaction products were thawed quickly in water at room temperature and separated on 10% native PAGE minigels in TBE buffer. Gels were washed in distilled water, mounted on Whatman 3 MM paper, wrapped in plastic and exposed to a phosphor screen that was scanned on a Typhoon 9400 laser imager (GE Healthcare). Scanned gels were analyzed using ImageQuant TL v2005 software (e.g. for Figure 2B and C). For all quantitations of helicase assays, the small % background of unannealed radiolabeled primer in the ‘No CMG’ lane was subtracted from the % unwound at each time point.