Cyclin-A-CDK2-mediated phosphorylation of CIZ1 blocks replisome formation and initiation of mammalian DNA replication.

CIZ1 is a nuclear matrix protein that cooperates with cyclin A2 (encoded by CCNA2) and CDK2 to promote mammalian DNA replication. We show here that cyclin-A-CDK2 also negatively regulates CIZ1 activity by phosphorylation at threonines 144, 192 and 293. Phosphomimetic mutants do not promote DNA replication in cell-free and cell-based assays, and also have a dominant-negative effect on replisome formation at the level of PCNA recruitment. Phosphorylation blocks direct interaction with cyclin-A-CDK2 and recruitment of endogenous cyclin A to the nuclear matrix. In contrast, phosphomimetic CIZ1 retains the ability to bind to the nuclear matrix, and its interaction with CDC6 is not affected. Phospho-T192-specific antibodies confirm that CIZ1 is phosphorylated during S phase and G2, and show that phosphorylation at this site occurs at post-initiation concentrations of cyclin-A-CDK2. Taken together, the data suggest that CIZ1 is a kinase sensor that promotes initiation of DNA replication at low kinase levels, when in a hypophosphorylated state that is permissive for cyclin-A-CDK2 interaction and delivery to licensed origins, but blocks delivery at higher kinase levels when it is phosphorylated.

Ciz1 cooperates with cyclin-A-CDK2 to activate mammalian DNA replication in vitro.

Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A-CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order.