APC/CCdh1-dependent proteolysis of USP1 regulates the response to UV-mediated DNA damage

Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome^Cdh1 (APC/C^Cdh1), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C^Cdh1. USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C^Cdh1 inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C^Cdh1 in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry.     

New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress

Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C^Cdh1 is activated upon severe replication stress. Activation of APC/C^Cdh1 prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C^Cdh1 activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C^Cdh1 activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C^Cdh1 function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.     

Damaged DNA-binding Protein 1 (DDB1) Interacts with Cdh1 and Modulates the Function of APC/CCdh1

APC/C^Cdh1 plays a key role in mitotic exit and has essential targets in the G₁ phase; however, these mechanisms are poorly understood. In this report, we provide evidence that damaged DNA-binding protein 1 (DDB1) is capable of binding the WD40 domains of Cdh1, but not of Cdc20, through its BPA and BPC domains. Moreover, cells lacking DDB1 exhibit markedly elevated levels of the protein substrates of APC/C^Cdh1. Depletion of DDB1 in mitotic cells significantly delays mitotic exit, which demonstrates that the interaction between DDB1 and Cdh1 plays a critical role in regulating APC/C^Cdh1 activity. However, cells depleted of Cdh1 demonstrated no change in the UV-induced degradation of Cdt1, the main function of DDB1 as an E3 ligase. Strikingly, the APC/C^Cdh1 substrate levels are normal in cell knockdowns of Cul4A and Cul4B, which, along with DDB1, form an E3 ligase complex. This finding indicates that DDB1 modulates the function of APC/C^Cdh1 in a manner independent on the Cul4-DDB1 complex. Our results suggest that DDB1 may functionally regulate mitotic exit by modulating APC/C^Cdh1 activity. This study reveals that there may be cross-talk among DDB1, Cdh1, and Skp2 in the control of cell cycle division.     

Residual Cdk1/2 activity after DNA damage promotes senescence

In response to DNA damage, a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity persists after DNA damage until terminal cell cycle exit. This low level of Cdk activity not only allows cell cycle progression, but also promotes cell cycle exit at a decision point in G2 phase. We find that residual Cdk1/2 activity is required for efficient p21 production, allowing for nuclear sequestration of Cyclin B1, subsequent APC/C^Cdh1‐dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle enforces senescence in the presence of DNA damage, ensuring a robust response when most needed.     

N Terminus of CtIP Is Critical for Homologous Recombination-mediated Double-strand Break Repair

DNA double-strand breaks (DSBs) represent one of the most lethal types of DNA damage cells encounter. CtIP (also known as RBBP8) acts together with the MRN (MRE11-RAD50-NBS1) complex to promote DNA end resection and the generation of single-stranded DNA, which is critically important for homologous recombination repair. However, it is not yet clear exactly how CtIP participates in this process. Here, we demonstrate that besides the known conserved C terminus, the N terminus of CtIP protein is also required in DSB end resection and DNA damage-induced G₂/M checkpoint control. We further show that both termini of CtIP can interact with the MRN complex and that the N terminus of CtIP, especially residues 22–45, binds to MRN and plays a critical role in targeting CtIP to sites of DNA breaks. Collectively, our results highlight the importance of the N terminus of CtIP in directing its localization and function in DSB repair.     

The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1‐βTrCP2‐Cdc25A‐mediated mitotic entry to ensure neurogenesis

Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase βTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of βTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1‐deficient neuroprogenitors in vivo. MCPH1 itself is degraded by APC/C^Cdh1, but not APC/C^Cdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1‐knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin‐proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development.     

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C^Cdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C^Cdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.     

Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C^Cdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1^m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C^Cdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.     

The ubiquitin proteasome system — Implications for cell cycle control and the targeted treatment of cancer

Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin–proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (anaphase promoting complex or cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/C^Cdc20) and establishes a stable G1 phase (APC/C^Cdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Fructokinase is required for carbon partitioning to cellulose in aspen wood

DNA damage checkpoints delay mitotic cell‐cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant‐specific cyclin‐dependent kinase (CDK) that is required for the G₂/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G₂ arrest gives cells time to repair damaged DNA. G₂ arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down‐regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis.