Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts

Abstract

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.

Involvement of CUL4 Ubiquitin E3 Ligases in Regulating CDK Inhibitors Dacapo/p27Kip1 and Cyclin E Degradation

“…21 CDT1 is also degraded in S phase in mammalian cells and such an event can be reproduced in Xenopus egg extracts in which CDT1 was found to undergo ubiquitin-dependent proteolysis once DNA replication starts. 37,38 In C. elegans, loss of CUL4 stabilizes CDT1 in S phase and causes the accumulation of polyploid nuclei containing 100C DNA content. 39 It is possible that CUL4 may also regulate the proteolysis of Dacapo or p27 in similar processes in Drosophila or human cells.…”

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing

“…In animal cells, overexpression of Cdt1 induces DNA re-replication (19,20,(32)(33)(34)(35), DNA fragmentation (36), chromosomal instability (37), and ATM/ATRdependent checkpoint activation (20,36). We therefore tested in U2OS cells wtCdt1 and the same two inactive mutants as in the Xenopus experiments for their ability to induce DNA rereplication, chromosomal damage, and checkpoint activation.…”

“…However, in the Xenopus early embryo, less than half of the endogenous Geminin is degraded at the exit from mitosis, while the remainder becomes inactivated and incapable of inhibiting Cdt1 (16)(17)(18). The remaining Geminin gets reactivated in the next cell cycle, through a mechanism that is not well understood, but is dependent on the nuclear import of Geminin, which restores its ability to block Cdt1 (16,19,20). Cell-cycle differences in Geminin's ability to inhibit Cdt1 has also been inferred from observations in other organisms (21)(22)(23)(24).…”

An ATR- and BRCA1-Mediated Fanconi Anemia Pathway Is Required for Activating the G2/M Checkpoint and DNA Damage Repair upon Rereplication

“…Overexpression of Cdt1 in human cells results in rereplication (43), and increased expression of Drosophila melanogaster Cdt1 (Dup) in diploid cells is sufficient to induce polyploidization (40). In Xenopus laevis egg extract, addition of Cdt1 after one round of DNA replication causes rereplication (2). Failure to degrade Cdt1 in S phase causes rereplication in Caenorhabditis elegans, Xenopus, and human cells (16,35,49).…”