In eukaryotes, replication licensing is achieved through sequential loading of several replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs). DNA replication initiates in pace with the rise of CDK (Cyclin-dependent kinase) and DDK (Dbf4p-dependent kinase) levels, which concertedly activate replication origins by phosphorylating Sld2p/3p and Mcm4p, respectively, to facilitate helicase activation and recruitment of DNA polymerases. Meanwhile, unscheduled replication licensing is prevented by CDK through inhibitory phosphorylations of multiple initiation proteins including pre-RC components Orc2p, Orc6p, Cdc6p and Mcm3p. On the other hand, CDK inactivation during mitotic exit promotes pre-RC formation for DNA replication in the next cell cycle...[ Read more ]

In eukaryotes, replication licensing is achieved through sequential loading of several replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs). DNA replication initiates in pace with the rise of CDK (Cyclin-dependent kinase) and DDK (Dbf4p-dependent kinase) levels, which concertedly activate replication origins by phosphorylating Sld2p/3p and Mcm4p, respectively, to facilitate helicase activation and recruitment of DNA polymerases. Meanwhile, unscheduled replication licensing is prevented by CDK through inhibitory phosphorylations of multiple initiation proteins including pre-RC components Orc2p, Orc6p, Cdc6p and Mcm3p. On the other hand, CDK inactivation during mitotic exit promotes pre-RC formation for DNA replication in the next cell cycle. However, the essentiality of removing both the activating and inhibitory phosphorylations on the replication proteins and the acting phosphatase(s) remain elusive. In this study, it is shown in budding yeast that Cdc14p dephosphorylates Sld2p/3p in early anaphase and Orc2p, Orc6p, Cdc6p as well as Mcm3p in late anaphase to promote pre-RC assembly. Based on these sequential dephosphorylations of distinct groups of replication factors, a two-step model is proposed in which replication origins are reprogrammed to their G1-like state by first extinguishing the origin-activating potential associated with the phosphorylated forms of Sld2p/3p, before resetting the competence for replication licensing by dephosphorylating pre-RC components.

Upon anaphase onset, mutual activation between separase and Cdc14p ensures the switch-like cohesion loss and hence near-synchrony separation of sister chromatids. Importantly, since Dbf4p is also an inhibitor of mitotic exit, and because I found that Cdc14p dephosphorylates and likely regulates Dbf4p stability, I propose an interlocked double positive feedback loop that operates at anaphase onset to advance robust degradation of Dbf4p and hence DDK inactivation as early as chromosomes segregation begins.