Replication-dependent Destruction of Cdt1 Limits DNA Replication to a Single Round Per Cell Cycle in Xenopus Egg Extracts

Overview

Journal Genes Dev

Specialty Molecular Biology

Date 2004 Dec 16

PMID 15598982

Citations 103

Authors

Emily E Arias

Johannes C Walter

Affiliations

Soon will be listed here.

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 alpha, 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.

Citing Articles

Single-molecule imaging reveals the mechanism of bidirectional replication initiation in metazoa.

Terui R, Berger S, Sambel L, Song D, Chistol G Cell. 2024; 187(15):3992-4009.e25.

PMID: 38866019 PMC: 11283366. DOI: 10.1016/j.cell.2024.05.024.

The cohesin modifier ESCO2 is stable during DNA replication.

Jevitt A, Rankin B, Chen J, Rankin S Chromosome Res. 2023; 31(1):6.

PMID: 36708487 PMC: 9884251. DOI: 10.1007/s10577-023-09711-1.

CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis.

Ratnayeke N, Baris Y, Chung M, Yeeles J, Meyer T Mol Cell. 2023; 83(1):26-42.e13.

PMID: 36608667 PMC: 7614657. DOI: 10.1016/j.molcel.2022.12.004.

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions.

Reusswig K, Bittmann J, Peritore M, Courtes M, Pardo B, Wierer M Nat Commun. 2022; 13(1):7014.

PMID: 36400763 PMC: 9674678. DOI: 10.1038/s41467-022-34379-2.

Disruption of Toxoplasma gondii-Induced Host Cell DNA Replication Is Dependent on Contact Inhibition and Host Cell Type.

Pierre-Louis E, Etheridge M, de Paula Baptista R, Khan A, Chasen N, Etheridge R mSphere. 2022; 7(3):e0016022.

PMID: 35587658 PMC: 9241542. DOI: 10.1128/msphere.00160-22.

References

Higa L, Mihaylov I, Banks D, Zheng J, Zhang H . Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat Cell Biol. 2003; 5(11):1008-15. DOI: 10.1038/ncb1061. View

Bates S, Ryan K, Phillips A, Vousden K . Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression. Oncogene. 1998; 17(13):1691-703. DOI: 10.1038/sj.onc.1202104. View

Oehlmann M, Score A, Blow J . The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation. J Cell Biol. 2004; 165(2):181-90. PMC: 2172031. DOI: 10.1083/jcb.200311044. View

Sugimoto N, Tatsumi Y, Tsurumi T, Matsukage A, Kiyono T, Nishitani H . Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding. J Biol Chem. 2004; 279(19):19691-7. DOI: 10.1074/jbc.M313175200. View

Melixetian M, Ballabeni A, Masiero L, Gasparini P, Zamponi R, Bartek J . Loss of Geminin induces rereplication in the presence of functional p53. J Cell Biol. 2004; 165(4):473-82. PMC: 2172361. DOI: 10.1083/jcb.200403106. View

Kondo T, Kobayashi M, Tanaka J, Yokoyama A, Suzuki S, Kato N . Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex. J Biol Chem. 2004; 279(26):27315-9. DOI: 10.1074/jbc.M314023200. View

Ekholm-Reed S, Mendez J, Tedesco D, Zetterberg A, Stillman B, Reed S . Deregulation of cyclin E in human cells interferes with prereplication complex assembly. J Cell Biol. 2004; 165(6):789-800. PMC: 2172392. DOI: 10.1083/jcb.200404092. View

Nishitani H, Lygerou Z, Nishimoto T . Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region. J Biol Chem. 2004; 279(29):30807-16. DOI: 10.1074/jbc.M312644200. View

Nguyen V, Co C, Irie K, Li J . Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7. Curr Biol. 2000; 10(4):195-205. DOI: 10.1016/s0960-9822(00)00337-7. View

10.

Prokhorova T, Mowrer K, Gilbert C, Walter J . DNA replication of mitotic chromatin in Xenopus egg extracts. Proc Natl Acad Sci U S A. 2003; 100(23):13241-6. PMC: 263766. DOI: 10.1073/pnas.2336104100. View

11.

Li A, Blow J . Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination. Nat Cell Biol. 2004; 6(3):260-7. PMC: 2691133. DOI: 10.1038/ncb1100. View

12.

Maiorano D, Rul W, Mechali M . Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis. Exp Cell Res. 2004; 295(1):138-49. DOI: 10.1016/j.yexcr.2003.11.018. View

13.

Liu E, Li X, Yan F, Zhao Q, Wu X . Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation. J Biol Chem. 2004; 279(17):17283-8. DOI: 10.1074/jbc.C300549200. View

14.

Zhu W, Chen Y, Dutta A . Rereplication by depletion of geminin is seen regardless of p53 status and activates a G2/M checkpoint. Mol Cell Biol. 2004; 24(16):7140-50. PMC: 479725. DOI: 10.1128/MCB.24.16.7140-7150.2004. View

15.

Thomer M, May N, Aggarwal B, Kwok G, Calvi B . Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2. Development. 2004; 131(19):4807-18. DOI: 10.1242/dev.01348. View

16.

Pacek M, Walter J . A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication. EMBO J. 2004; 23(18):3667-76. PMC: 517609. DOI: 10.1038/sj.emboj.7600369. View

17.

Newport J . A role for Ran-GTP and Crm1 in blocking re-replication. Cell. 2004; 119(1):145. DOI: 10.1016/j.cell.2004.09.021. View

18.

Shechter D, Ying C, Gautier J . DNA unwinding is an Mcm complex-dependent and ATP hydrolysis-dependent process. J Biol Chem. 2004; 279(44):45586-93. DOI: 10.1074/jbc.M407772200. View

19.

Lohka M, Masui Y . Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983; 220(4598):719-21. DOI: 10.1126/science.6601299. View

20.

Hershko A, Heller H . Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates. Biochem Biophys Res Commun. 1985; 128(3):1079-86. DOI: 10.1016/0006-291x(85)91050-2. View