The stimulation of unwinding by Mcm10 observed in Figure 2 could be attributable to more efficient loading of CMG onto the substrate, faster unwinding, and/or greater processivity of unwinding. To distinguish among these possibilities, we compared CMG unwinding of a fork with a longer, 160 bp duplex region to that of the fork with a 50 bp duplex (as in Figure 2) in the presence and absence of Mcm10 (Figure 3). The two substrates have identical 3’ and 5’ ssDNA tails, so loading of CMG onto the forks should be the same and any differences in unwinding should be attributable to differences in CMG unwinding activity over the different lengths of duplex. For these experiments, CMG was pre-incubated with the DNA substrate for 10’ followed by addition of ATP ± Mcm10 to start the reaction (reaction scheme in Figure 3A). In the absence of Mcm10, substantial differences were observed in unwinding of the two substrates that suggest limited processivity of the helicase, consistent with reports of low processivity by Drosophila and human CMG (Kang et al., 2012; Moyer et al., 2006). CMG unwound only 3% of the longer 160 bp duplex fork in 10’ (Figure 3B lanes 1–10 and graph in Figure 3D) compared to 24% for the shorter 50 bp duplex fork at 10’ (Figure 3C). Even after 30’ only 8% of the 160 bp duplex was unwound by CMG indicating that the difference in activity between the two substrates is attributable to low processivity of CMG rather than simply the additional time it takes to unwind the longer substrate.