APC/CCdh1 controls CtIP stability during the cell cycle and in response to DNA damage

Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C^Cdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage‐induced G₂ arrest. However, it is currently unknown whether APC/C^Cdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA‐damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA‐end resection factor, as a novel APC/C^Cdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G₁ and after DNA damage in G₂. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA‐end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/C^Cdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle‐ and DNA damage‐dependent manner.     

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.     

Anaphase-promoting complex/cyclosome-cdh1 mediates the ubiquitination and degradation of TRB3

We have recently demonstrated that TRB3, a novel endoplasmic reticulum (ER) stress-inducible protein, is induced by CHOP and ATF4 to regulate their function and ER stress-induced cell death; however, the regulation of TRB3 function has not been well characterized. Here we demonstrate that TRB3 is an unstable protein regulated by the ubiquitin-proteasome system. The carboxyl-terminal domain of TRB3 is necessary for protein degradation, and in this region, we found the typical D-box motif, which is a critical sequence for the anaphase-promoting complex/cyclosome (APC/C) dependent proteolysis. TRB3 proteins were stabilized by deletion of its D-box motif and interacted with APC/C coactivator proteins, Cdc20 and Cdh1. The expression level of TRB3 protein is down-regulated by over-expression of Cdh1 but not by that of Cdc20. In addition, knockdown of Cdh1 enhanced the endogenous TRB3 expression level and suppressed its ubiquitination level. These results suggest that APC/C^Cdh1 is involved in ubiquitination and down-regulating the stability of TRB3 protein.     

Proteolysis of Rad17 by Cdh1/APC regulates checkpoint termination and recovery from genotoxic stress

Recent studies have shown a critical function for the ubiquitin‐proteasome system (UPS) in regulating the signalling network for DNA damage responses and DNA repair. To search for new UPS targets in the DNA damage signalling pathway, we have carried out a non‐biased assay to identify fast‐turnover proteins induced by various types of genotoxic stress. This endeavour led to the identification of Rad17 as a protein exhibiting a distinctive pattern of upregulation followed by subsequent degradation after exposure to UV radiation in human primary cells. Our characterization showed that UV‐induced Rad17 oscillation is mediated by Cdh1/APC, a ubiquitin‐protein ligase. Studies using a degradation‐resistant Rad17 mutant demonstrated that Rad17 stabilization prevents the termination of checkpoint signalling, which in turn attenuates the cellular re‐entry into cell‐cycle progression. The findings provide an insight into how the proteolysis of Rad17 by Cdh1/APC regulates the termination of checkpoint signalling and the recovery from genotoxic stress.     

BubR1 acetylation at prometaphase is required for modulating APC/C activity and timing of mitosis

Regulation of BubR1 is central to the control of APC/C activity. We have found that BubR1 forms a complex with PCAF and is acetylated at lysine 250. Using mass spectrometry and acetylated BubR1-specific antibodies, we have confirmed that BubR1 acetylation occurs at prometaphase. Importantly, BubR1 acetylation was required for checkpoint function, through the inhibition of ubiquitin-dependent BubR1 degradation. BubR1 degradation began before the onset of anaphase. It was noted that the pre-anaphase degradation was regulated by BubR1 acetylation. Degradation of an acetylation-mimetic form, BubR1–K250Q, was inhibited and chromosome segregation in cells expressing BubR1–K250Q was markedly delayed. By contrast, the acetylation-deficient mutant, BubR1–K250R, was unstable, and mitosis was accelerated in BubR1–K250R-expressing cells. Furthermore, we found that APC/C–Cdc20 was responsible for BubR1 degradation during mitosis. On the basis of our collective results, we propose that the acetylation status of BubR1 is a molecular switch that converts BubR1 from an inhibitor to a substrate of the APC/C complex, thus providing an efficient way to modulate APC/C activity and mitotic timing.     

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

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Chronic restraint stress disturbs meiotic resumption through APC/C-mediated cyclin B1 excessive degradation in mouse oocytes

Psychological stress, which exerts detrimental effects on human reproduction, may compromise the meiotic competence of oocytes. Meiotic resumption, germinal vesicle breakdown (GVBD), is the first milestone to confer meiotic competence to oocytes. In the practice of assisted reproductive technology (ART), the timing for GVBD is associated with the rates of cleavage and blastocyst formation. However, whether chronic stress compromises oocyte competence by influencing GVBD and the underlying mechanisms are unclear. In the present study, a chronic restraint stress (CRS) mouse model was used to investigate the effects of stress on oocyte meiotic resumption, as well as the mechanisms. Following a 4-week chronic restraint stress in female mice, the percentage of abnormal bipolar spindles increased and indicated compromised oocyte competence in the CRS group. Furthermore, we identified a decreased percentage of GVBD and prolonged time of GVBD in the CRS mouse oocytes compared with the control group. CRS simultaneously reduced the expression of cyclin B1 (CCNB1), which represents a regulatory subunit of M-phase/mature promoting factor (MPF). However, MG132, an inhibitor of anaphase-promoting complex/cyclosome (APC/C), could rescue the prolonged time of GVBD and increase the expression level of CCNB1 of oocytes from the CRS mice. Collectively, our results demonstrated that stress disturbed meiotic resumption through APC/C-mediated CCNB1 degradation, thus providing a novel understanding for stress-related oocyte quality decline; moreover, it may provide a non-invasive approach to select high-quality gametes and novel targets for molecular therapy to treat stress-related female infertility.     

Phosphorylation of Skp2 regulated by CDK2 and Cdc14B protects it from degradation by APC(Cdh1) in G1 phase

The p27(Kip1) ubiquitin ligase receptor Skp2 is often overexpressed in human tumours and displays oncogenic properties. The activity of SCF(Skp2) is regulated by the APC(Cdh1), which targets Skp2 for degradation. Here we show that Skp2 phosphorylation on Ser64/Ser72 positively regulates its function in vivo. Phosphorylation of Ser64, and to a lesser extent Ser72, stabilizes Skp2 by interfering with its association with Cdh1, without affecting intrinsic ligase activity. Cyclin-dependent kinase (CDK)2-mediated phosphorylation of Skp2 on Ser64 allows its expression in mid-G1 phase, even in the presence of active APC(Cdh1). Reciprocally, dephosphorylation of Skp2 by the mitotic phosphatase Cdc14B at the M --> G1 transition promotes its degradation by APC(Cdh1). Importantly, lowering the levels of Cdc14B accelerates cell cycle progression from mitosis to S phase in an Skp2-dependent manner, demonstrating epistatic relationship of Cdc14B and Skp2 in the regulation of G1 length. Thus, our results reveal that reversible phosphorylation plays a key role in the timing of Skp2 expression in the cell cycle.     

Molecular mechanism of activation of human Cdc7 kinase: bipartite interaction with Dbf4/activator of S phase kinase (ASK) activation subunit stimulates ATP binding and substrate recognition

Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I-III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448-457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432-574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the "minimum" Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.     

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.     

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop

The anaphase‐promoting complex/cyclosome (APC/C), a multi‐subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin‐dependent kinase 1 (Cdk1) promotes Cdc20 co‐activator loading in mitosis to form active APC/C‐Cdc20. However, detailed phospho‐regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo‐like kinase (Plx1) and PP2A‐B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1‐loop⁵⁰⁰) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1‐loop⁵⁰⁰ in a phosphorylation‐dependent manner and promotes the formation of APC/C‐Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A‐B56 is recruited to the Apc1‐loop⁵⁰⁰ and differentially promotes dissociation of Plx1 and PP2A‐B56 through dephosphorylation of Plx1‐binding sites. Stable Plx1 binding, which prevents PP2A‐B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1‐loop⁵⁰⁰ is controlled by distant Apc3‐loop phosphorylation. Our study suggests that phosphorylation‐dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.     

Premature Cdk1/Cdc5/Mus81 pathway activation induces aberrant replication and deleterious crossover

The error‐free DNA damage tolerance (DDT) pathway is crucial for replication completion and genome integrity. Mechanistically, this process is driven by a switch of templates accompanied by sister chromatid junction (SCJ) formation. Here, we asked if DDT intermediate processing is temporarily regulated, and what impact such regulation may have on genome stability. We find that persistent DDT recombination intermediates are largely resolved before anaphase through a G2/M damage checkpoint‐independent, but Cdk1/Cdc5‐dependent pathway that proceeds via a previously described Mus81‐Mms4‐activating phosphorylation. The Sgs1‐Top3‐ and Mus81‐Mms4‐dependent resolution pathways occupy different temporal windows in relation to replication, with the Mus81‐Mms4 pathway being restricted to late G2/M. Premature activation of the Cdk1/Cdc5/Mus81 pathway, achieved here with phosphomimetic Mms4 variants as well as in S‐phase checkpoint‐deficient genetic backgrounds, induces crossover‐associated chromosome translocations and precocious processing of damage‐bypass SCJ intermediates. Taken together, our results underscore the importance of uncoupling error‐free versus erroneous recombination intermediate processing pathways during replication, and establish a new paradigm for the role of the DNA damage response in regulating genome integrity by controlling crossover timing.