In eukaryotes, DNA replication takes place during the S-phase of the cell cycle. The cell cycle control system, consisting of a network of regulatory proteins, governs the progression of the cell cycle.

The S-phase cyclin-dependent kinase, or S-Cdks, are enzyme complexes involved in cell cycle control during S-phase. This control system ensures that every nucleotide in the genome is copied only once during replication.

DNA replication begins at the origins of replication, present at multiple locations in the chromosomes. Before the initiation of replication, multiprotein complexes called origin recognition complexes bind to the DNA and serve as docking sites for other proteins.

During early G1-phase of the cell cycle, the regulatory proteins Cdc6 and Cdt1 bind to the ORCs, and aid in the assembly of a set of proteins called the MCM proteins into inactive ring complexes on the adjacent DNA, thereby forming large multiprotein complexes called the pre-replicative complexes or pre-RCs. MCMs function as DNA helicases for replication.

At the onset of S-phase, S-Cdks are activated and trigger origin firing, or initiation of DNA replication, by phosphorylating specific initiator proteins. Phosphorylated initiator proteins promote the recruitment of helicase activator complexes. These complexes activate the DNA helicases and recruit the DNA polymerase, leading to replication.

S-Cdks not only initiate replication but also prevent re-replication from occurring at the same origin.

S-Cdks phosphorylate the Cdc6 and Cdt1 proteins, promoting their release from the ORCs, leading to degradation, and the disassembly of the pre-RCs.

After DNA replication, when the helicases disengage from the DNA strand, the S-Cdks phosphorylate the helicases, triggering their export from the nucleus.

This cell cycle control mechanism involving S-Cdks prevents re-initiation of replication, thereby ensuring that the DNA replication occurs only once per cell cycle.