The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication 
to trigger a cascade of protein associations that activate the Mcm2-7 
replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase 
preferentially targets chromatin-associated Mcm2-7 complexes that are assembled 
on the DNA during prereplicative complex (pre-RC) formation. Here we address the 
mechanisms that control the specificity of DDK action. We show that 
incorporation of Mcm2-7 into the pre-RC increased the level and changes the 
specificity of DDK phosphorylation of this complex. In the context of the 
pre-RC, DDK preferentially targets a conformationally distinct subpopulation of 
Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting 
requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent 
manner. Importantly, we find that DDK association with and phosphorylation of 
origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. Our 
findings provide insights into the mechanisms that ensure that DDK action is 
spatially and temporally restricted to the origin-bound Mcm2-7 complexes that 
will drive replication fork movement during S phase and suggest new mechanisms 
to regulate origin activity.