The Mcm2-7 complex has in vitro helicase activity.

Helicases unwind duplex DNA ahead of the polymerases at the replication fork. However, the identity of the eukaryotic replicative helicase has been controversial; in vivo studies implicate the ring-shaped heterohexameric Mcm2-7 complex, although only a specific subset of Mcm subunits (Mcm467) unwind DNA in vitro. To address this discrepancy, we have compared both Mcm assemblies and find that they differ in their linear single-stranded DNA association rate and their ability to bind circular single-stranded DNA. These differences depend upon the Mcm2/5 interface, which we hypothesize serves as an ATP-dependent "gate" within Mcm2-7. Importantly, we find that reaction conditions that putatively close the Mcm2-7 "gate" reconstitute Mcm2-7 helicase activity. Unlike Mcm467, Mcm2-7 helicase activity is strongly anion dependent. Our results show that purified Mcm2-7 acts as a helicase, provides functional evidence of a Mcm2/5 gate, and lays the foundation for future mechanistic studies of this critical factor.