It has been reported that the 3′-extension can be unwound by the archaeal Mcm homohexamer but not by fission and budding yeast Mcm complexes (14,26,27). We have constructed 3′-extension substrates containing poly(dT), poly(dA), poly(dC) and poly(dG) 3′-tails in order to examine whether mouse Mcm4/6/7 can displace these substrates. We first found that Mcm4/6/7 binds to dT-, dC- and dG-3′-extension with similar affinity, and to dA-3′-extension with much lower affinity (Figure 5A). This is consistent with the previous results that Mcm4/6/7 binds to 50mer oligo-dT, -dC and -dG, but not to a 50mer oligo-dA (13). We found that dT-tailed 3′-extension was displaced efficiently by Mcm 4/6/7 while almost no or very little activity was detected on other substrates (Figure 5B and C). This is in sharp contrast to yeast Mcm4/6/7 helicases which are not capable of unwinding 3′-extension substrates. Furthermore, dT-tailed 5′-extension, to which Mcm4/6/7 could bind, was not displaced by the Mcm helicase. The addition of the 5′-tail to dT-tailed 3′-extension further stimulated the displacement (Figure 5D), confirming the previous report that the fork structures facilitate the helicase action. Thus, these results further strengthen our conclusion that the mammalian Mcm helicase is generally activated by the thymine-stretch on the 3′-single-stranded tail.