Cul4 E3 ubiquitin ligases contain the cullin 4 scaffold and the triple beta propeller Ddb1 adaptor protein, but few substrate receptors have been identified. Here, we identify 18 Ddb1- and Cul4-associated factors (DCAFs), including 14 containing WD40 repeats. DCAFs interact with multiple surfaces on Ddb1, and the interaction of WD40-containing DCAFs with Ddb1 requires a conserved "WDXR" motif. DCAF2/Cdt2, which is related to S. pombe Cdt2, functions in Xenopus egg extracts and human cells to destroy the replication licensing protein Cdt1 in S phase and after DNA damage. Depletion of human Cdt2 causes rereplication and checkpoint activation. In Xenopus, Cdt2 is recruited to replication forks via Cdt1 and PCNA, where Cdt1 ubiquitylation occurs. These studies uncover diverse substrate receptors for Cul4 and identify Cdt2 as a conserved component of the Cul4-Ddb1 E3 that is essential to destroy Cdt1 and ensure proper cell cycle regulation of DNA replication.

In eukaryotic cells, prereplication complexes (pre-RCs) are assembled on chromatin in the G1 phase, rendering origins of DNA replication competent to initiate DNA synthesis. When DNA replication commences in S phase, pre-RCs are disassembled, and multiple initiations from the same origin do not occur because new rounds of pre-RC assembly are inhibited. In most experimental organisms, multiple mechanisms that prevent pre-RC assembly have now been identified, and rereplication within the same cell cycle can be induced through defined perturbations of these mechanisms. This review summarizes the diverse array of inhibitory pathways used by different organisms to prevent pre-RC assembly, and focuses on the challenge of understanding how in any one cell type, various mechanisms cooperate to strictly enforce once per cell cycle regulation of DNA replication.The ability of eukaryotic cells to duplicate vast amounts of genetic information quickly and accurately before each cell division is an awe-inspiring product of evolution. To achieve rapid replication, cells use a parallel processing approach in which multiple replisomes copy DNA simultaneously. A dramatic example of this strategy occurs during the early cleavage divisions of the frog Xenopus laevis, in which DNA replication initiates from ∼300,000 sites, called origins, which are spaced ∼10 kb apart (Blow 2001). As a result, 3 billion base pairs of DNA are duplicated within minutes. All eukaryotic organisms use a similar strategy, and the number of origins employed is generally correlated to the genome size and the length of the cell cycle. To ensure that DNA replication is not only fast, but accurate, it is essential that no segment of the chromosome be duplicated more than once. Indeed, reinitiation from even a single origin within the same cell cycle may cause genome instability. Thus, the ability of cells to restrict DNA replication to a single round per cell cycle is a fundamental requirement of cell proliferation and long-term survival. The two-state model for cell cycle regulation of DNA replicationEarly insights into the regulation of eukaryotic DNA replication came from cell fusion experiments (Rao and Johnson 1970), which showed that union of an S-phase cell with a G1 cell accelerates the rate at which the latter enters S phase. In contrast, G2 cells are refractory to this stimulation. These results suggested that the initiation of DNA synthesis requires a positive, diffusible S-phasepromoting activity, and that G1 but not G2-phase cells are competent to respond to this signal. Building on these observations and on their own experiments in Xenopus egg extracts, Blow and Laskey (1988) developed the "licensing" model, in which a licensing factor that is required for replication initiation binds to chromatin in G1. At the G1/S transition, the factor is inactivated, and it cannot be replenished until cells pass through mitosis. These ideas were extended by in vivo footprinting experiments in yeast that showed that origins of replication alternate between two...

Ubiquitin-mediated proteolysis of the replication licensing factor Cdt1 (Cdc10-dependent transcript 1) in S phase is a key mechanism that limits DNA replication to a single round per cell cycle in metazoans. In Xenopus egg extracts, Cdt1 is destroyed on chromatin during DNA replication. Here, we report that replication-dependent proteolysis of Cdt1 requires its interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric processivity factor for DNA polymerases. Cdt1 binds to PCNA through a consensus PCNA-interaction motif that is conserved in Cdt1 of all metazoans, and removal of PCNA from egg extracts inhibits replication-dependent Cdt1 destruction. Mutation of the PCNA-interaction motif yields a stabilized Cdt1 protein that induces re-replication. DDB1, a component of the Cul4 E3 ubiquitin ligase that mediates human Cdt1 proteolysis in response to DNA damage, is also required for replication-dependent Cdt1 destruction. Cdt1 and DDB1 interact in extracts, and DDB1 chromatin loading is dependent on the binding of Cdt1 to PCNA, which indicates that PCNA docking activates the pre-formed Cdt1-Cul4(DDB1) ligase complex. Thus, PCNA functions as a platform for Cdt1 destruction, ensuring efficient and temporally restricted inactivation of a key cell-cycle regulator.

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In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. Here, we show that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase ␣, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. Our results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase.[Keywords: DNA replication; Cdt1; geminin; Cdk; proteolysis; Xenopus] Supplemental material is available at http://www.genesdev.org.