Eukaryotic origins of replication are selected by loading a head-to-head double 
hexamer of the Mcm2-7 replicative helicase around origin DNA. Cdt1 plays an 
essential but transient role during this event; however, its mechanism of action 
is unknown. Through analysis of Cdt1 mutations, we demonstrate that Cdt1 
performs multiple functions during helicase loading. The C-terminus of Cdt1 
binds Mcm2-7, and this interaction is required for efficient origin recruitment 
of both proteins. We show that origin recognition complex (ORC) and Cdc6 recruit 
multiple Cdt1 molecules to the origin during helicase loading, and disruption of 
this multi-Cdt1 intermediate prevents helicase loading. Although dispensable for 
loading Mcm2-7 double hexamers that are topologically linked to DNA, the 
essential N-terminal domain of Cdt1 is required to load Mcm2-7 complexes that 
are competent for association with the Cdc45 and GINS helicase-activating 
proteins and replication initiation. Our data support a model in which 
origin-bound ORC and Cdc6 recruit two Cdt1 molecules to initiate double-hexamer 
formation prior to helicase loading and demonstrate that Cdt1 influences the 
replication competence of loaded Mcm2-7 helicases.