Robust progression through the cell-division cycle depends on the precisely ordered phosphorylation of hundreds of different proteins by cyclin-dependent kinases (CDKs) and other kinases. The order of CDK substrate phosphorylation depends on rising CDK activity, coupled with variations in substrate affinities for different CDK-cyclin complexes and the opposing phosphatases 1–4. Here, we address the ordering of substrate phosphorylation by a second major cell-cycle kinase, Cdc7-Dbf4 or Dbf4-dependent kinase (DDK). The primary function of DDK is to initiate DNA replication by phosphorylating the Mcm2-7 replicative helicase 5–7. DDK also phosphorylates the cohesin acetyltransferase Eco1 8. Sequential phosphorylations of Eco1 by CDK, DDK, and Mck1 create a phosphodegron that is recognized by the ubiquitin ligase SCFCdc4. DDK, despite being activated in early S phase, does not phosphorylate Eco1 to trigger its degradation until late S phase 8. DDK associates with docking sites on loaded Mcm double hexamers at unfired replication origins 9, 10. We hypothesized that these docking interactions sequester limiting amounts of DDK, delaying Eco1 phosphorylation by DDK until replication is complete. Consistent with this hypothesis, we find that overproduction of DDK leads to premature Eco1 degradation. Eco1 degradation also occurs prematurely if Mcm complex loading at origins is prevented by depletion of Cdc6, and Eco1 is stabilized if loaded Mcm complexes are prevented from firing by a Cdc45 mutant. We propose that the timing of Eco1 phosphorylation, and potentially that of other DDK substrates, is determined in part by sequestration of DDK at unfired replication origins during S phase. •Dbf4-Cdc7 (DDK) phosphorylates Eco1 in late S phase, triggering Eco1 degradation•DDK activity toward Eco1 is limiting during S phase•DDK is sequestered during S phase by Mcm complexes at unfired replication origins Seoane and Morgan explore the mechanism underlying the cell-cycle timing of Eco1 phosphorylation, and they present evidence that the protein kinase Dbf4-Cdc7 does not phosphorylate Eco1 until late S phase because the kinase is sequestered during S phase by Mcm complexes at unfired replication origins.