Eukaryotic cells license each DNA replication origin during G1 phase by 
assembling a prereplication complex that contains a Mcm2-7 (minichromosome 
maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer 
forms the core of a replicative DNA helicase. However, the mechanisms of origin 
licensing and helicase activation are poorly understood. The helicase loaders 
ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication 
origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the 
second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not 
well understood. Here, structural evidence for intermediates consisting of an 
ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, 
which together provide new insights into DNA licensing. Detailed structural 
analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two 
hexamers are interlocked and misaligned along the DNA axis and lack ATP 
hydrolysis activity that is essential for DNA helicase activity. Moreover, we 
show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates 
a new protein interaction surface that creates a multisubunit-binding site for 
an S-phase protein kinase that is known to activate DNA replication. The data 
suggest how the double hexamer is assembled and how helicase activity is 
regulated during DNA licensing, with implications for cell cycle control of DNA 
replication and genome stability.