In order to examine more precisely the interaction and helicase actions with forked substrates, Y-fork substrates containing a nascent leading strand (A-fork[3′]), nascent lagging strand (A-fork[5′]), or both of them (A-fork[3′,5′]), as well as 5′-extension or 3′-extension structures were constructed. Mcm4/6/7 bound to A-fork[3′] or A-fork[5′] with affinity similar to that of a simple Y-fork (Figure 4A and data not shown). In contrast, PriA helicase, used as a control, bound to A-fork[3′] more efficiently than to A-fork[5′], as reported previously (23,25). Mcm4/6/7 bound also to 5′-extension and 3′-extension, albeit with slightly reduced affinity compared to A-fork[3′] and A-fork[5′] (Figure 4A). Mcm4/6/7 bound to A-fork[3′,5′] with much lower affinity, and the complex gave rise to a single band. These findings indicate that the Mcm4/6/7 complex is loaded onto DNA preferentially and efficiently through single-stranded region, regardless of the presence of 3′- or 5′-terminus.