In order to distinguish these two possibilities, we designed a new T-fork substrate containing inosine (I) residue instead of guanosine residue. The thermal stability of the I:C base pair is lower than that of the G:C base pair in duplex DNA due to loss of one hydrogen bond and is even lower than A:T base pair (31). We constructed the T-tailed fork substrates containing the (GCC:CGG)10, (GCC:CIG)10 or (GAA:CTT)10 duplex DNA segment (Figure 8E). Mcm4/6/7 hardly displaced the 31 nt long duplex of GCC:CGG repeats, whereas the T-fork/(GAA:CTT)10 was displaced efficiently, as described above. T-fork/(GCC:CIG)10 was displaced with efficiency much greater than that of T-fork/(GCC:CGG)10, indicating that the thermostability of the duplex segment, not the lack of AT base pair, is responsible for inability of Mcm4/6/7 to displace the duplex.