Claspin: Timing the Cell Cycle Arrest When the Genome is Damaged

DNA damage checkpoints maintain genomic integrity by delaying cell cycle progression in response to genotoxic stress and stalled replication forks. One central pathway in the checkpoint response is the ATR-Chk1 pathway, in which, upon DNA damage, ATR phosphorylates and activates the effector kinase Chk1. This process depends on the adaptor protein Claspin that bridges ATR and Chk1. Once the damage is repaired, this pathway must somehow be switched off to allow the cell to continue the cell division process, an event known as checkpoint recovery. Polo-like kinase 1 (Plk1) plays a central role during checkpoint recovery. Interestingly, the Xenopus homologue of Plk1, Plx1, is able to bind and phosphorylate Claspin, releasing it from DNA and thereby contributing to Chk1 inactivation. Moreover, it was recently demonstrated that Claspin levels are controlled by proteasomal degradation, and this is regulated by Plk1. Importantly, Plk1-mediated proteosomal degradation of Claspin appears to be essential for checkpoint recovery. Here we review these recent findings and discuss the mechanisms of checkpoint regulation by Claspin.     

Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress

We show that Claspin, an adaptor protein required for Chk1 activation, becomes degraded at the onset of mitosis. Claspin degradation was triggered by its interaction with, and ubiquitylation by, the SCFbetaTrCP ubiquitin ligase. This interaction was phosphorylation dependent and required the activity of the Plk1 kinase and the integrity of a betaTrCP recognition motif (phosphodegron) in the N terminus of Claspin. Uncoupling of Claspin from betaTrCP by mutating the conserved serines in Claspin's phosphodegron or by knocking down betaTrCP stabilized Claspin in mitosis, impaired Chk1 dephosphorylation, and delayed G2/M transition during recovery from cell cycle arrest imposed by DNA damage or replication stress. Moreover, the inability to degrade Claspin allowed partial reactivation of Chk1 in cells exposed to DNA damage after passing the G2/M transition. Our data suggest that degradation of Claspin facilitates timely reversal of the checkpoint response and delineates the period permissive for Chk1 activation during cell cycle progression.     

The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint

In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here, we show that, in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/C(Cdh1), with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/C(Cdh1) reactivation in DNA-damaged G2 cells, Claspin, which we show to be an APC/C(Cdh1) substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint.     

Ectopic Expression of Plk1 Leads to Activation of the Spindle Checkpoint

Polo-like kinase 1 (Plk1), the best characterized member of the mammalian polo-like kinase family, is well regulated throughout the cell cycle, and is inhibited following DNA damage. Chk1 plays a key role in the response to DNA damage. We recently reported that Chk1 is required for mitotic progression through negative regulation of Plk1. Here, we report the phenotypes of cultured cells upon ectopic expression of various forms of Plk1. Epitopic expression of Plk1 led to mitotic arrest, whereas co-expression of Chk1 could release this mitotic block. Moreover, the Plk1 expression-induced mitotic block was also released by inactivation of the spindle-assembly checkpoint.     

SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response

During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1 by ATR. We found that during recovery from the DNA replication checkpoint response, Claspin is degraded in a betaTrCP-dependent manner. In vivo, Claspin is phosphorylated in a canonical DSGxxS degron sequence, which is typical of betaTrCP substrates. Phosphorylation of Claspin is mediated by Plk1 and is essential for binding to betaTrCP. In vitro ubiquitylation of Claspin requires betaTrCP, Plk1, and an intact DSGxxS degron. Significantly, expression of a stable Claspin mutant unable to bind betaTrCP prolongs the activation of Chk1, thereby attenuating the recovery from the DNA replication stress response and significantly delaying entry into mitosis. Thus, the SCFbetaTrCP-dependent degradation of Claspin is necessary for the efficient and timely termination of the DNA replication checkpoint. Importantly, in response to DNA damage in G2, Claspin proteolysis is inhibited to allow the prompt reestablishment of the checkpoint.     

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1

In vertebrates, the checkpoint-regulatory kinase Chk1 mediates cell-cycle arrest in response to a block in DNA replication or to DNA damaged by ultraviolet radiation. The activation of Chk1 depends on both Claspin and the upstream regulatory kinase ATR. Claspin is a large acidic protein that becomes phosphorylated and binds to Chk1 in the presence of checkpoint-inducing DNA templates in Xenopus egg extracts. Here we identify, by means of deletion analysis, a region of Claspin of 57 amino acids that is both necessary and sufficient for binding to Xenopus Chk1. This Chk1-binding domain contains two highly conserved repeats of approximately ten amino acids. A serine residue in each repeat (serine 864 and serine 895) undergoes phosphorylation during a checkpoint response. A mutant of Claspin containing non-phosphorylatable amino acids at positions 864 and 895 cannot bind to Chk1 and is unable to mediate its activation. Our results indicate that two phosphopeptide motifs in Claspin are essential for checkpoint signalling.     

Checkpoint Adaptation and Recovery: Back with Polo after the Break

S. cerevisiae cells that are unable to repair a double strand break ultimatelyescape the DNA damage checkpoint arrest and enter mitosis. This process called'adaptation' depends on functional Cdc5, a Polo-like kinase, and was long thoughtto be limited to single-cell organisms. However, the recent finding that Xenopusextracts can adapt to a long-lasting stall in DNA replication indicates thatcheckpoint adaptation does also occur in multicellular organisms. Interestingly, theXenopus Polo-like kinase (Plx1) plays an important role in this adaptation. To addto this, data from our laboratory have shown that the human Polo-like kinase (Plk1)is also required for cell cycle re-entry following a DNA damage-induced arrest. Buthere, Plk1 was shown to be required for bona-fide checkpoint recovery, rather thanadaptation. That is, Plk1 is required to restart the cell cycle once all of the damage isrepaired and checkpoint signaling is turned off. While the target of Plx1 duringadaptation is a component of the checkpoint machinery (Claspin), the target of Plk1during recovery turns out to be a mitotic regulator (Wee1). Here, we discuss some ofthe remarkable similarities and subtle differences in the molecular mechanisms thatcontrol checkpoint adaptation and recovery, and the role of Polo-like kinasestherein.     

Regulation of Polo-like kinase 1 by DNA damage in mitosis. Inhibition of mitotic PLK-1 by protein phosphatase 2A

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. In addition to Plk1 functions in cell cycle, Plk1 is involved in DNA damage check-point in G2 phase. Normally, ataxia telangiectasia-mutated kinase (ATM) is a key enzyme involved in G2 phase cell cycle arrest following DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in a ATM/ATR-dependent manner. However, it is still unclear how Plk1 is regulated in response to DNA damage in mitosis in which Plk1 is already activated. Here, we show that treatment of mitotic cells with doxorubicin and gamma-irradiation inhibits Plk1 activity through dephosphorylation of Plk1, and cells were arrested in G2 phase. Treatments of the phosphatase inhibitors and siRNA experiments suggested that PP2A pathway might be involved in regulating mitotic Plk1 activity in mitotic DNA damage. Finally, we propose a novel pathway, which is connected between ATM/ATR/Chk and protein phosphatase-Plk1 in DNA damage response in mitosis.     

Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response

BACKGROUND: Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators. RESULTS: By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest. CONCLUSIONS: Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.     

Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Less is known about the mechanisms that allow resumption of the cell cycle once checkpoint signaling is silenced. Here we show that while in undamaged cells several redundant pathways can promote the onset of mitosis, this redundancy is lost in cells recovering from a DNA damage-induced arrest. We demonstrate that Plk1 is crucial for mitotic entry following recovery from DNA damage. However, Plk1 is no longer required in cells depleted of Wee1, and we could show that Plk1 is involved in the degradation of Wee1 at the onset of mitosis. Thus, our data show that the cell cycle machinery is reset in response to DNA damage and that cells become critically dependent on Plk1-mediated degradation of Wee1 for their recovery.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

Structure meets function--centrosomes, genome maintenance and the DNA damage response

Centrosomes are cytoplasmic organelles playing a fundamental role in organizing both the interphase cytoskeleton and the bipolar mitotic spindle. In addition, the centrosome has recently come into focus as part of the network that integrates cell cycle arrest and repair signals in response to genotoxic stress--the DNA damage response. One important mediator of this response, the checkpoint kinase Chk1, has been shown to negatively regulate the G(2)/M transition via its centrosomal localization. Moreover, there is growing evidence that a centrosome inactivation checkpoint exists, which utilizes DNA damage-induced centrosome fragmentation or amplification to provoke a "mitotic catastrophe" and eliminate damaged cells. Candidate regulators of this centrosomal checkpoint include the checkpoint kinase Chk2 and its upstream regulators ATM and ATR. In addition, a growing number of other proteins have been implicated in centrosomal regulation of the DNA damage response, e.g. the tumor suppressor p53, the breast cancer susceptibility gene product BRCA1 and mitotic regulators such as Aurora A, Nek2 and the Polo-like kinases Plk1 and Plk3. However, many missing links and discrepancies between different model systems remain.     

Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ)

Polo-like kinase 1 (Plk1) is an instrumental kinase that modulates many aspects of the cell cycle. Previous investigations have indicated that Plk1 is a target of the DNA damage response, and Plk1 inhibition is dependent on ATM/ATR and Chk1. But the exact mechanism remains elusive. In a proteomic screen to identify Chk1-interacting proteins, we found that myosin phosphatase targeting protein 1 (MYPT1) was present in the immunocomplex. MYPT1 is phosphorylated by CDK1, thus recruiting protein phosphatase 1β (PP1cβ) to dephosphorylate and inactivate Plk1. Here we identified that Chk1 directly interacts with MYPT1 and preferentially phosphorylates MYPT1 at Ser20, which is essential for MYPT1-PP1cβ interaction and subsequent Plk1 dephosphorylation. Phosphorylation of Ser20 is abolished during mitotic damage when Chk1 is inhibited. The degradation of MYPT1 is also regulated by Chk1 phosphorylation. Our results thus unveil the underlying machinery that attenuates Plk1 activity during mitotic damage through Chk1-induced phosphorylation of MYPT1.     

Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage

The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.     

Mitotic DNA damage response: Polo-like Kinase-1 is dephosphorylated through ATM-Chk1 pathway

DNA damage during the cell division cycle can activate ATM/ATR and their downstream kinases that are involved in the checkpoint pathway, and cell growth is halted until damage is repaired. As a result of DNA damage induced in mitotic cells by doxorubicin treatment, cells accumulate in a G2-like phase, not in mitosis. Under these conditions, two mitosis-specific kinases, Cdk1 and Plk1, are inhibited by inhibitory phosphorylation and dephosphorylation, respectively. G2-specific phosphorylation of Cdc25 was increased during incubation after mitotic DNA damage. Inhibition of Plk1 through dephosphorylation was dependent on ATM/Chk1 activity. Depleted expression of ATM and Chk1 was achieved using small hairpin RNA (shRNA) plasmid constructs. In this condition, damaged mitotic cells did not accumulated in a G2-like stage, and entered into G1 phase without delay. Protein phosphatase 2A was responsible for dephosphorylation of mitotic Plk1 in response to DNA damage. In knockdown of PP2A catalytic subunits, Plk1 was not dephosphorylated, but rather degraded in response to DNA damage, and cells did not accumulate in G2-like phase. The effect of ATM/Chk1 inhibition was counteracted by overexpression of PP2A, indicated that PP2A may function as a downstream target of ATM/Chk1 at a mitotic DNA damage checkpoint, or may have a dominant effect on ATM/Chk1 function at this checkpoint. Finally, we have shown that negative regulation of Plk1 by dephosphorylation is important to cell accumulation in G2-like phase at the mitotic DNA damage checkpoint, and that this ATM/Chk1/PP2A pathway independent on p53 is a novel mechanism of cellular response to mitotic DNA damage.     

Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint

The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Δmik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.     

USP7 counteracts SCFβTrCP- but not APCCdh1-mediated proteolysis of Claspin

Claspin is an adaptor protein that facilitates the ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1, a key effector kinase in the DNA damage response. Efficient termination of Chk1 signaling in mitosis and during checkpoint recovery requires SCF^βTrCP-dependent destruction of Claspin. Here, we identify the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Claspin stability. Claspin and USP7 interact in vivo, and USP7 is required to maintain steady-state levels of Claspin. Furthermore, USP7-mediated deubiquitylation markedly prolongs the half-life of Claspin, which in turn increases the magnitude and duration of Chk1 phosphorylation in response to genotoxic stress. Finally, we find that in addition to the M phase–specific, SCF^βTrCP-mediated degradation, Claspin is destabilized by the anaphase-promoting complex (APC) and thus remains unstable in G1. Importantly, we demonstrate that USP7 specifically opposes the SCF^βTrCP- but not APC^Cdh1-mediated degradation of Claspin. Thus, Claspin turnover is controlled by multiple ubiquitylation and deubiquitylation activities, which together provide a flexible means to regulate the ATR–Chk1 pathway.