RESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Cromatina/química , Reações Cruzadas , Modelos BiológicosRESUMO
In the last decade, extraordinary advances in our understanding of the initiation step of eukaryotic DNA replication have been achieved. Many factors required for replication initiation have been identified, and an elegant model to explain how DNA replication is restricted to a single round per cell cycle has emerged. Of the many experimental approaches used to study DNA replication, egg extracts from Xenopus laevis are among the most powerful, since they recapitulate a complete round of cell-cycle regulated chromosomal DNA replication in vitro. In this review, we discuss current models for how DNA replication is initiated and regulated in Xenopus eggs, and we highlight similarities and differences seen between this and the other most common experimental organisms, yeast and humans.
Assuntos
Replicação do DNA , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Animais , Ciclo Celular , Núcleo Celular/metabolismo , Sistema Livre de Células , Cromatina/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Origem de Replicação , XenopusRESUMO
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.
Assuntos
Replicação do DNA , Células Eucarióticas/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Humanos , Modelos Biológicos , Origem de Replicação , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Processamento de Proteína Pós-Traducional , Fase S , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , Dano ao DNA , Replicação do DNA/genética , Fase G2 , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Reprodutibilidade dos Testes , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Neoplasias/fisiologia , Fragmentos de Peptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/fisiologia , Ubiquitina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/efeitos da radiação , Linhagem Celular , Proteínas Culina/efeitos da radiação , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/efeitos da radiação , Células HCT116 , Humanos , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/efeitos da radiação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/efeitos da radiação , Antígeno Nuclear de Célula em Proliferação/efeitos da radiação , Fase S/genética , Fase S/fisiologia , Fase S/efeitos da radiação , Proteínas Ligases SKP Culina F-Box/fisiologia , Raios UltravioletaRESUMO
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.