(A) The helicase reaction in Figure 5B (CMG + Mcm10) was repeated with the radiolabel on the biotinylated lagging strand and in the presence of free biotin as a trap for displaced streptavidin (See Materials and methods for details). Unwound strands are shifted in the gel when bound to streptavidin (product +SA) compared to unbound (product – SA). Lanes 2–4 are a control in the absence of biotin trap showing that all of the unwound products migrate in the gel shifted position. The biotin trap is present in the experiment in lanes 5–7. A shift in migration of the unwound products indicates displacement of streptavidin by CMG-Mcm10, but the result shows that almost all the unwound DNA retained the streptavidin. (B) The experiment in (A) was repeated with a substrate in which the dual biotin blocks were on the radiolabeled leading strand. Also see Figure 6—figure supplement 1.