Figure 6.

Multiple concerted pathways prevent DNA re-replication in mammals. Once mammalian cells have entered S phase, re-replication of DNA that has already been replicated is prevented by inactivation of Orc1, Cdc6, and Cdt1 by direct Cdk2•CcnA dependent phosphorylation. In addition, Cdc6-P is localized to the cytoplasm, and both Orc1-P and Cdt1-P are substrates for the ubiquitin ligase CRL1•Skp2. Cdt1 that is not phosphorylated, but associated with PCNA and DNA, is targeted for degradation by the CRL4•Cdt2 ubiquitin ligase. PCNA is the eukaryotic sliding clamp protein that allows DNA polymerases-δ and -γ to synthesize DNA continuously without falling off the primer–template. Binding of Geminin also inhibits Cdt1, and Orc1 is also inhibited directly by Cdk1•CcnA phosphorylation, events that are required during G2 and early M phase.