Entry into S phase is defined by the unwinding of origins of DNA replication. The molecular events leading to this unwinding are best understood in S. cerevisiae. G₁/S phase CDKs play an essential role in this process. They turn off the machinery that degrades S phase cyclins during exit from mitosis and G₁, and they induce the degradation of a CKI that inhibits S phase CDKs.

One of the important substrates of the G₁/S phase cyclin-CDK complexes is Cdh1. During late anaphase, this substrate targeting factor directs APC/C to ubiquitinylate substrate proteins, including S phase and mitotic cyclins, marking them for proteolysis by proteasomes. APC/C^Cdh1 remains active throughout G₁, preventing the premature accumulation of S phase and mitotic cyclins. Phosphorylation of Cdh1 by G₁/S cyclin-CDKs causes it to dissociate from the APC/C complex, inhibiting further ubiquitinylation of S phase and mitotic cyclins during late G₁ (Figure 19-13). This inhibition, combined with the induced transcription of S phase cyclins during late G₁, allows S phase cyclins to accumulate as G₁/S cyclin-CDK levels rise. Later in the cell cycle, S phase and mitotic CDKs take over to maintain Cdh1 in the phosphorylated, and hence inactive, state. Only as mitotic CDKs decline and a protein phosphatase known as Cdc14 is activated are these inhibitory phosphates removed from Cdh1, leading to its reactivation. In mammalian cells, similar mechanisms are responsible for stabilizing S phase and mitotic cyclins, but the phosphatase(s) involved in dephosphorylation of Cdh1 have not been identified.

In S. cerevisiae, as S phase cyclin-CDK heterodimers accumulate in late G₁ following the inactivation of APC/C^Cdh1, they are immediately inactivated by binding of a CKI called Sic1 that is expressed late in mitosis and early in G₁ (Figure 19-14). Because Sic1 specifically inhibits S phase and M phase CDK complexes, but has no effect on the G₁ CDK and G₁/S phase CDK complexes, it functions as an S phase inhibitor. Initiation of DNA replication occurs when the Sic1 inhibitor is precipitously degraded following its ubiquitinylation by the SCF ubiquitin-protein ligase.

Degradation of Sic1 is induced by its phosphorylation by G₁/S phase CDKs (see Figure 19-14). It must be phosphorylated at no fewer than six sites, which are relatively poor substrates for the G₁/S phase CDKs, before it is bound sufficiently well by SCF to be ubiquitinylated. These multiple, poor G₁/S phase CDK phosphorylation sites lead to an ultrasensitive, switch-like response in Sic1 degradation and hence precipitous activation of S phase CDKs (Figure 19-15). If Sic1 were inactivated following the phosphorylation of a single site, Sic1 molecules would start to get phosphorylated as soon as the levels of G₁/S phase CDK activity begin to rise, leading to a gradual decrease in Sic1 levels. In contrast, because several sites need to be phosphorylated, at low levels of G₁/S phase CDK activity only a few sites are phosphorylated and Sic1 is not destroyed. Only when G₁/S phase CDK levels are high is Sic1 sufficiently phosphorylated at multiple sites to target it for degradation. Thus Sic1 degradation occurs only when G₁/S phase CDK activity has reached its peak and virtually all other G₁/S phase CDK substrates have been phosphorylated.

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Once Sic1 is degraded, the S phase cyclin-CDK complexes induce DNA replication by, as we will see shortly, phosphorylating several proteins involved in activating the replicative helicases. This mechanism for activating the S phase cyclin-CDK complexes—that is, inhibiting them as the cyclins are synthesized and then precipitously degrading the inhibitor—permits the sudden initiation of replication at large numbers of replication origins. An obvious advantage of proteolysis for controlling passage through this critical point in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed in one direction through the cycle.

Entry into S phase in metazoan cells is regulated by a mechanism similar to that in budding yeast. Like Sic1, the CKI p27 prevents the premature activation of S phase CDKs during G₁. Unlike Sic1, however, this CKI inhibits both S phase CDKs and G₁/S phase CDKs and has additional cell cycle functions as well. For example, while inhibiting G₁/S phase CDKs and S phase CDKs, p27 helps assemble and hence activate G₁ CDKs. Like Sic1 in yeast, however, p27 is removed from cyclin-CDK complexes by ubiquitin-dependent protein degradation. Two pathways contribute to its degradation. Upon stimulation by mitogens, mitogen-activated protein kinases phosphorylate p27, promoting its export from the nucleus into the cytoplasm, where one of the cellular ubiquitin-protein ligases, KPC, is found. A second pathway, analogous to the one operating on Sic1, targets p27 for degradation at the G₁-S phase transition. As G₁/S phase CDKs and S phase CDKs reach high levels during late G₁ and early S phase, they begin to phosphorylate p27, targeting it for ubiquitinylation by SCF. Degradation of p27 causes activation of G₁/S phase and S phase CDKs. These kinases then initiate S phase by phosphorylating proteins important for the initiation of DNA replication.