Cdt1 forms a complex with MCM and activates its helicase activity

Mcm4/6/7 forms a complex possessing DNA helicase activity, suggesting that Mcm may be a central component for the replicative helicase. Although Cdt1 is known to be essential for loading of Mcm onto the chromatin, its precise role in pre-RC formation and replication initiation is unknown. Using purified proteins, we show that Cdt1 forms a complex with Mcm4/6/7, Mcm2/3/4/5/6/7 and Mcm2/4/6/7 in glycerol gradient fractionation through interacting with Mcm2 and Mcm4/6. In the glycerol gradient fractionation, Mcm4/6/7-Cdt1 forms a complex (speculated to be a (Mcm4/6/7)2-Cdt13 assembly) in the presence of ATP which is significantly larger than the Mcm4/6/7-Cdt1 complex generated in its absence. Furthermore, the DNA binding and helicase activities of Mcm4/6/7 are significantly stimulated by Cdt1 protein in vitro. We generated a Cdt1 mutant which fails to stimulate DNA binding and helicase activities of Mcm4/6/7. This mutant Cdt1 showed reduced interaction with Mcm and deficient in formation of the high molecular weight complex with Mcm. Thus, a productive interaction between Cdt1 and MCM appears to be essential for efficient loading of MCM onto template DNA as well as for efficient unwinding reaction.