The experiments were repeated in the presence of Mcm10 at a 2:1 ratio to CMG (as determined in Figure 2A–C). Surprisingly, in the presence of Mcm10 the unwinding curves of the two substrates were very similar, in contrast to experiments in the absence of Mcm10 (Figure 3B and C, lanes 11–19 and graph in Figure 3D). This result suggests that Mcm10 enhances the processivity of CMG and possibly also stimulates the rate of CMG unwinding. Examination of the first 5 min of unwinding in the presence of Mcm10 shows a short delay in the appearance of products using the 160 bp duplex compared to the 50 bp duplex (Figure 3E). We took advantage of this delayed appearance of the longer products to design an experiment that approximates the average rate of unwinding by CMG-Mcm10 (Figure 3—figure supplement 1). CMG was pre-incubated with an equimolar mixture of the two different length substrates and then the reaction was started by the addition of ATP and Mcm10. The substrates and products migrate at distinguishable positions in a native PAGE gel allowing us to observe the appearance of both products in the same reaction. As shown in Figure 3—figure supplement 1, appearance of unwound 160 bp duplex products was delayed by ~46 s compared to the 50 bp duplex, indicating an average rate of unwinding of 2.4 bp/s over the additional 110 bp. The final extent of unwinding is similar for both substrates, so the delay reflects a difference in time of unwinding the two DNAs due to their difference in length rather than a difference in helicase activity on the two substrates.