Search tips
Search criteria

Results 1-25 (1398040)

Clipboard (0)

Related Articles

1.  A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint 
PLoS Biology  2010;8(1):e1000287.
A combined computational and biochemical approach reveals how mitotic kinases allow cell division to proceed in the presence of DNA damage.
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.
Author Summary
DNA is constantly damaged both by factors outside our bodies (such as ultraviolet rays from sunlight) and by factors from within (such as reactive oxygen species produced during metabolism). DNA damage can lead to malfunctioning of genes, and persistent DNA damage can result in developmental disorders or the development of cancer. To ensure proper DNA repair, cells are equipped with an evolutionarily conserved DNA damage checkpoint, which stops proliferation and activates DNA repair mechanisms. Intriguingly, this DNA damage checkpoint responds to DNA damage throughout the cell cycle, except during mitosis. In this work, we have addressed how cells dismantle their DNA damage checkpoint during mitosis to allow cell division to proceed even if there is damaged DNA present. Using the observation that kinases phosphorylate their substrates on evolutionarily conserved, kinase-specific sequence motifs, we have used a combined computational and experimental approach to predict and verify key proteins involved in mitotic checkpoint inactivation. We show that the checkpoint scaffold protein 53BP1 is phosphorylated by the mitotic kinases Cdk1 and Polo-like kinase-1 (Plk1). Furthermore, we find that Plk1 can inactivate the checkpoint kinase Chk2, which is downstream of 53BP1. Plk1 is shown to be a key mediator of mitotic checkpoint inactivation, as cells that cannot activate Plk1 fail to properly dismantle the DNA damage checkpoint during mitosis and instead show DNA damage-induced Chk2 kinase activation. Two related papers, published in PLoS Biology (Vidanes et al., doi:10.1371/journal.pbio.1000286) and PLoS Genetics (Donnianni et al., doi:10.1371/journal.pgen.1000763), similarly investigate the phenomenon of DNA damage checkpoint silencing.
PMCID: PMC2811157  PMID: 20126263
2.  Caenorhabditis elegans Cyclin B3 Is Required for Multiple Mitotic Processes Including Alleviation of a Spindle Checkpoint–Dependent Block in Anaphase Chromosome Segregation 
PLoS Genetics  2010;6(11):e1001218.
The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3–depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3–dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC–dependent block in anaphase chromosome segregation.
Author Summary
Every time a cell divides in two, the genetic material, DNA, is copied; each copied chromosome is referred to as a pair of sister chromatids. Each chromatid must be cleanly separated from its sister so that each daughter cell inherits the same DNA complement as the starting cell. The mitotic spindle is a cellular machine that physically separates the sister chromatids from one another. The chromatids are attached to the spindle at kinetochores, which are structures built at specific sites (centromeres) on each chromatid. The cell monitors the attachment of each chromatid and blocks their separation until they are all properly attached. This process is called the spindle assembly checkpoint (SAC). Here we report that loss of an evolutionarily conserved cell cycle regulator, Cyclin B3/CYB-3, results in an unusual and strikingly persistent SAC–dependent delay in sister chromatid separation. Furthermore, CYB-3 promotes the activity of a cellular motor, dynein, in this and other mitotic processes. Altogether, our results indicate that Cyclin B3 genetically interacts with mitotic dynein and is absolutely required to satisfy a SAC–dependent inhibition in sister chromatid separation.
PMCID: PMC2991249  PMID: 21124864
3.  DNA Damage Activates the SAC in an ATM/ATR-Dependent Manner, Independently of the Kinetochore 
PLoS Genetics  2008;4(2):e1000015.
The DNA damage checkpoint and the spindle assembly checkpoint (SAC) are two important regulatory mechanisms that respond to different lesions. The DNA damage checkpoint detects DNA damage, initiates protein kinase cascades, and inhibits the cell cycle. The SAC relies on kinetochore-dependent assembly of protein complexes to inhibit mitosis when chromosomes are detached from the spindle. The two checkpoints are thought to function independently. Here we show that yeast cells lacking the DNA damage checkpoint arrest prior to anaphase in response to low doses of the DNA damaging agent methyl methane sulfonate (MMS). The arrest requires the SAC proteins Mad1, Mad2, Mad3, Bub1, and Bub3 and works through Cdc20 and Pds1 but unlike the normal SAC, does not require a functional kinetochore. Mec1 (ATR) and Tel1 (ATM) are also required, independently of Chk1 and Rad53, suggesting that Mec1 and Tel1 inhibit anaphase in response to DNA damage by utilizing SAC proteins. Our results demonstrate cross-talk between the two checkpoints and suggest that assembling inhibitory complexes of SAC proteins at unattached kinetochores is not obligatory for their inhibitory activity. Furthermore, our results suggest that there are novel, important targets of ATM and ATR for cell cycle regulation.
Author Summary
Genome integrity is assured, in part, by regulatory systems called “checkpoints” that assure that cells do not improperly progress through the cell cycle. The DNA damage checkpoint assesses the status of DNA replication and inhibits cell cycle progression when the cell makes mistakes in DNA replication or when the cell has been assaulted by a DNA damaging agent from the environment. The checkpoint allows the cell time to repair the DNA and then permits the cell cycle to resume. There is a separate “spindle checkpoint” that monitors whether chromosomes are properly attached to the spindle and if so, allows cells to proceed through mitosis. The DNA damage checkpoint and the spindle checkpoint assure that daughter cells receive the correct number of chromosomes that are identical in DNA sequence. Here we show that the two checkpoints are not independent but that they cooperate to restrict mitotic progression in the face of DNA damage. We show that the spindle checkpoint can be induced by DNA damage and that there is a novel kinetochore independent mechanism to activate the spindle checkpoint proteins. In addition, we implicate the ATM and ATR kinases as kinetochore-independent activators of the spindle checkpoint.
PMCID: PMC2265443  PMID: 18454191
4.  Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae 
Cell Cycle  2010;9(16):3353-3363.
The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. the checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. the ira1Δ ira2Δ recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery and implicates the Ras signaling pathway as an important regulator of mitotic events.
PMCID: PMC3041168  PMID: 20716966
DNA damage checkpoint; Ras signaling; budding yeast; cAMP-dependent protein kinase; anaphase promoting complex; neurofibromatosis type 1
5.  CDC5 Inhibits the Hyperphosphorylation of the Checkpoint Kinase Rad53, Leading to Checkpoint Adaptation 
PLoS Biology  2010;8(1):e1000286.
The mechanistic role of the yeast kinase CDC5, in allowing cells to adapt to the presence of irreparable DNA damage and continue to divide, is revealed.
The Saccharomyces cerevisiae polo-like kinase Cdc5 promotes adaptation to the DNA damage checkpoint, in addition to its numerous roles in mitotic progression. The process of adaptation occurs when cells are presented with persistent or irreparable DNA damage and escape the cell-cycle arrest imposed by the DNA damage checkpoint. However, the precise mechanism of adaptation remains unknown. We report here that CDC5 is dose-dependent for adaptation and that its overexpression promotes faster adaptation, indicating that high levels of Cdc5 modulate the ability of the checkpoint to inhibit the downstream cell-cycle machinery. To pinpoint the step in the checkpoint pathway at which Cdc5 acts, we overexpressed CDC5 from the GAL1 promoter in damaged cells and examined key steps in checkpoint activation individually. Cdc5 overproduction appeared to have little effect on the early steps leading to Rad53 activation. The checkpoint sensors, Ddc1 (a member of the 9-1-1 complex) and Ddc2 (a member of the Ddc2/Mec1 complex), properly localized to damage sites. Mec1 appeared to be active, since the Rad9 adaptor retained its Mec1 phosphorylation. Moreover, the damage-induced interaction between phosphorylated Rad9 and Rad53 remained intact. In contrast, Rad53 hyperphosphorylation was significantly reduced, consistent with the observation that cell-cycle arrest is lost during adaptation. Thus, we conclude Cdc5 acts to attenuate the DNA damage checkpoint through loss of Rad53 hyperphosphorylation to allow cells to adapt to DNA damage. Polo-like kinase homologs have been shown to inhibit the ability of Claspin to facilitate the activation of downstream checkpoint kinases, suggesting that this function is conserved in vertebrates.
Author Summary
Cellular surveillance mechanisms, termed checkpoints, have evolved to recognize the presence of DNA damage, halt cell division, and promote repair. The purpose of these checkpoints is to prevent the next generation of cells from inheriting a damaged genome. However, after futile attempts at repair over several hours of growth arrest, yeast cells eventually adapt and continue with cell division despite the presence of persistent DNA lesions. This process of adaptation employs CDC5, a kinase that also has essential roles in promoting cell division in the absence of DNA damage. We found that increasing levels of Cdc5 promote adaptation by suppressing the hyperphosphorylation of the checkpoint kinase Rad53, which in turn suppresses the DNA damage checkpoint and relieves cell division arrest. Intriguingly, overexpression of PLK1, the human homolog of CDC5, has been reported in various tumor types and has been linked to poor prognosis. Therefore, understanding the mechanism of adaptation in yeast may provide valuable insight into the role of PLK1 overexpression in tumor progression. Two related papers, published in PLoS Biology (van Vugt et al., doi:10.1371/journal.pbio.1000287) and PLoS Genetics (Donnianni et al., doi:10.1371/journal.pgen.1000763), similarly investigate the phenomenon of checkpoint adaptation.
PMCID: PMC2811153  PMID: 20126259
6.  Checkpoints couple transcription network oscillator dynamics to cell-cycle progression 
Genome Biology  2014;15(9):446.
The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior.
Here we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression.
Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0446-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4180952  PMID: 25200947
7.  CDK1 plays an important role in the maintenance of pluripotency and genomic stability in human pluripotent stem cells 
Cell Death & Disease  2014;5(11):e1508-.
Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are characterised by an unusual and tightly regulated cell cycle that has been shown to be important for the maintenance of a pluripotent phenotype. Cyclin-dependant kinase 1 (CDK1) is a key player in cell cycle regulation and particularly mitosis; however, its role has not been studied previously in hESC and hiPSC. To investigate the impacts of CDK1 downregulation, we performed RNA interference studies which in addition to expected mitotic deficiencies revealed a large range of additional phenotypes related to maintenance of pluripotency, ability to repair double strand breaks (DSBs) and commitment to apoptosis. Downregulation of CDK1 led to the loss of typical pluripotent stem cell morphology, downregulation of pluripotency markers and upregulation of a large number of differentiation markers. In addition, human pluripotent stem cells with reduced CDK1 expression accumulated a higher number of DSBs were unable to activate CHK2 expression and could not maintain G2/M arrest upon exposure to ionising radiation. CDK1 downregulation led to the accumulation of cells with abnormal numbers of mitotic organelles, multiple chromosomal abnormalities and polyploidy. Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process. On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation. Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.
PMCID: PMC4260724  PMID: 25375373
8.  Phosphorylation of the Bloom's Syndrome Helicase and Its Role in Recovery from S-Phase Arrest 
Molecular and Cellular Biology  2004;24(3):1279-1291.
Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3+ related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G2/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.
PMCID: PMC321429  PMID: 14729972
9.  Dot1-Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor 
PLoS Genetics  2013;9(1):e1003262.
During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along zip1 meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.
Author Summary
In sexually reproducing organisms, meiosis divides the number of chromosomes by half to generate gametes. Meiosis involves a series of interactions between maternal and paternal chromosomes leading to the exchange of genetic material by recombination. Completion of these processes is required for accurate distribution of chromosomes to the gametes. Meiotic cells possess quality-control mechanisms (checkpoints) to monitor those critical events. When failures occur, the checkpoint blocks meiotic progression to prevent the formation of aneuploid gametes. Genetic information is packaged into chromatin; histone modifications regulate multiple aspects of DNA metabolism to maintain genomic integrity. Dot1 is a conserved methyltransferase, responsible for histone H3 methylation at lysine 79, that is required for the meiotic recombination checkpoint. Here we decipher the molecular mechanism underlying Dot1 meiotic checkpoint function. We show that Dot1 catalytic activity correlates with the strength of the checkpoint response. By regulating Pch2 chromatin distribution, Dot1 controls localization of the chromosome axial component Hop1, which, in turn, contributes to activation of Mek1, the major effector kinase of the checkpoint. Our findings suggest that, in response to meiotic defects, the chromatin environment created by a constitutive histone mark orchestrates distribution of structural components of the chromosomes supporting activation of the meiotic checkpoint.
PMCID: PMC3561090  PMID: 23382701
10.  Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest 
Besides the well-understood DNA damage response via establishment of G2 checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re-entry. The aim of this study was to investigate the role of Chk1 in the recovery from G2 checkpoint arrest in HCT116 (human colorectal cancer) wt, p53–/– and p21–/– cell lines following H2O2 treatment. Firstly, DNA damage caused G2 checkpoint activation via Chk1. Secondly, overriding G2 checkpoint led to (i) mitotic slippage, cell cycle re-entry in G1 and subsequent G1 arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1-involved G2 arrest with respect to p53/p21WAF1: absence of either protein led to late G2 arrest instead of the classic G2 arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G2 arrest correlated with downstream senescence, but late G2 arrest led to mitotic catastrophe, although both cell cycle re-entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long-term DNA damage responses causing cell cycle re-entry. We propose that recovery from oxidative DNA damage-induced G2 arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21WAF1. The general relevance of Chk1 as an important determinant of recovery from G2 checkpoint arrest was verified in HT29 colorectal cancer cells.
PMCID: PMC3823197  PMID: 20716119
DNA damage; G2 checkpoint; recovery; checkpoint-kinase 1 (Chk1); senescence; mitotic catastrophe
11.  Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway 
PLoS Genetics  2010;6(1):e1000763.
Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.
Author Summary
Double strand DNA breaks (DSBs) are dangerous chromosomal lesions that can lead to genome rearrangements, genetic instability, and cancer if not accurately repaired. Eukaryotes activate a surveillance mechanism, called DNA damage checkpoint, to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to DSBs, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Budding yeast is a good model system to study checkpoint, and most of the factors involved in the DSBs response were originally characterized in this organism. Using the yeast Saccharomyces cerevisiae, we explored the functional role of polo kinase Cdc5 in regulating the DSB–induced checkpoint. Polo kinases have been previously involved in checkpoint inactivation in all the eukaryotes, and they are frequently overexpressed in cancer cells. We found that elevated levels of Cdc5 affect the cellular response to a DSB at different steps, altering DNA processing and overriding the signal triggered by checkpoint kinases. Our findings suggest that Cdc5 likely regulates multiple factors in response to a DSB and provide a rationale for a proteome-wide screening to identify targets of polo kinases in yeast and human cells. Such information may have a practical application to design specific molecular tools for cancer therapy. Two related papers published in PLoS Biology—by Vidanes et al., doi:10.1371/journal.pbio.1000286, and van Vugt et al., doi:10.1371/journal.pbio.1000287—similarly investigate the phenomenon of checkpoint adaptation/overriding.
PMCID: PMC2797610  PMID: 20098491
12.  Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1997;17(5):2723-2734.
Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.
PMCID: PMC232123  PMID: 9111343
13.  DNA Damage Response and Spindle Assembly Checkpoint Function throughout the Cell Cycle to Ensure Genomic Integrity 
PLoS Genetics  2015;11(4):e1005150.
Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.
Author Summary
Checkpoints are surveillance pathways that monitor and correct cellular errors to ensure that the genome is transmitted intact through cell division; defects in checkpoints lead to human disease such as cancer. Two major checkpoint pathways that have been extensively studied are the DNA damage response and the spindle assembly checkpoint. As their names imply, they have been thought to monitor distinct chromosomal events during the cell cycle. Here, we used C. elegans proliferating germ cells and human cells to investigate the role of these checkpoints when either DNA is damaged or the spindle is perturbed. We discovered that these checkpoints function together in response to these different perturbations to ensure genome integrity. Our studies have important implications for cancer treatments, as many cancer chemotherapies target one of these checkpoint pathways without consideration for the effect on the other pathway.
PMCID: PMC4405263  PMID: 25898113
14.  Cdk2 Is Required for p53-Independent G2/M Checkpoint Control 
PLoS Genetics  2010;6(2):e1000863.
The activation of phase-specific cyclin-dependent kinases (Cdks) is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G2/M checkpoint activation.
Author Summary
Metazoan cells contain multiple Cdks that regulate cell cycle progression. Recent studies have shown that mouse cells can grow normally with just Cdk1. The roles of the non-essential Cdks remain a fundamental question. In this study, we describe the generation and detailed characterization of CDK2-knockout human somatic cells. Our study demonstrates that Cdk2 is required for robust DNA damage checkpoint signaling. Loss of Cdk2 caused a marked deficiency in the G2/M arrest—a basic response to DNA damage—in cells that were also nullizygous for P53. We propose that the multiple Cdks present in metazoan cells provide a mechanism by which the cell cycle can be efficiently halted after DNA damage. Significantly, this study reveals a heretofore unrecognized dependence for Cdk2 in p53-deficient cancer cells.
PMCID: PMC2829054  PMID: 20195506
15.  Feedback between p21 and reactive oxygen production is necessary for cell senescence 
The sustained activation of CDKN1A (p21/Waf1/Cip1) by a DNA damage response induces mitochondrial dysfunction and reactive oxygen species (ROS) production via signalling through CDKN1A-GADD45A-MAPK14- GRB2-TGFBR2-TGFbeta in senescing primary human and mouse cells in vitro and in vivo.Enhanced ROS production in senescing cells generates additional DNA damage. Although this damage is repairable and transient, it elevates the average levels of DNA damage response permanently, thus forming a positive feedback loop.This loop is necessary and sufficient to maintain the stability of growth arrest until a ‘point of no return' is reached during establishment of senescence.
The phenomenon of cellular ‘senescence'—the permanent arrest of division in normally proliferating mammalian cells such as fibroblasts—is thought to be a central component of the ageing process. Senescence contributes both to age-related loss of tissue homeostasis, as the loss of division capacity leads to impaired cell renewal, and also to protect against cancer, because it acts to block the uncontrolled proliferation of cells that may give rise to a malignant tumour. Replicative senescence is triggered by uncapped telomeres or by ‘unrepairable' non-telomeric DNA damage. Both lesions initiate the same canonical DNA damage response (DDR) (d'Adda di Fagagna, 2008). This response is characterized by activation of sensor kinases (ATM/ATR, DNA-PK), formation of DNA damage foci containing activated H2A.X (γH2A.X) and ultimately induction of cell cycle arrest through activation of checkpoint proteins, notably p53 (TP53) and the CDK inhibitor p21 (CDKN1A). This signalling pathway continues to contribute actively to the stability of the G0 arrest in fully senescent cells long after induction of senescence (d'Adda di Fagagna et al, 2003). However, senescence is more complex than mere CDKI-mediated growth arrest. Senescent cells alter their expression of literally hundreds of genes (Shelton et al, 1999), prominent among these being pro-inflammatory secretory genes (Coppe et al, 2008) and marker genes for a retrograde response induced by mitochondrial dysfunction (Passos et al, 2007a).
There is a growing evidence that multiple mechanisms interact to underpin ageing at the cellular level (Kirkwood, 2005; Passos et al, 2007b) necessitating a systems biology approach if the complex mechanisms of ageing are to be understood (Kirkwood, 2008). With respect to cell senescence, the two major unanswered questions are (i) How does a DNA lesion that can be repaired, at least in principle, induce and maintain irreversible growth arrest? and (ii) How does a growth arrest trigger a completely different cellular phenotype as soon as it becomes irreversible?
To understand those questions, we performed a kinetic analysis of the establishment phase of senescence initiated by DNA damage or telomere dysfunction, focussing on pathways downstream of the classical DDR. Using an approach that combined (i) in-silico interactome analysis, (ii) functional target gene inhibition, (iii) stochastic modelling, and (iv) live cell microscopy, we identified a positive feedback loop between DDR and mitochondrial production of reactive oxygen species (ROS) as necessary and sufficient for long-term maintenance of growth arrest. Using pathway log likelihood scores calculated by a quantitative in-silico interactome analysis to guide siRNA and small molecule inhibition experiments, and using results of sequential and combined inhibition experiments to refine the predictions from the interactome analysis, we found that DDR triggered mitochondrial dysfunction leading to enhanced ROS activation through a linear signal transduction through TP53, CDKN1A, GADD45A, p38 (MAPK14), GRB2, TGFBR2 and TGFβ(Figure 2D). We hypothesized that these ROS stochastically generate novel DNA damage in the nucleus, thus forming a positive feedback loop contributing to the long-term maintenance of DDR (Figure 3A). First confirmation came from static inhibitor experiments as before, showing that nuclear DNA damage foci frequencies in senescent cells were reduced if feedback signalling was suppressed. To formally establish the existence of a feedback loop and its relevance for senescence, we used live cell microscopy in combination with quantitative modelling.
We transformed the conceptual model shown in Figure 3A into a stochastic mechanistic model of the DDR feedback loop by extending the previously published model of the TP53/Mdm2 circuit (Proctor and Gray, 2008) to include reactions for synthesis/activation and degradation/deactivation/repair of CDKN1A, GADD45, MAPK14, ROS and DNA damage. The model replicated very precisely the kinetic behaviour of activated TP53, CDKN1A, ROS and DNA damage foci after initiation of senescence by irradiation. Having established its concordance with the experimental data, the model was then used to predict the effects of intervening in the feedback loop. The model predicted that any intervention reducing ROS levels by about half would decrease average DNA damage foci frequencies from six to four foci/nucleus within about 15 h. It further predicted that this would be sufficient to reduce CDKN1A to basal levels continuously for at least 6 h in about 20% of the treated cells, thus allowing a significant fraction of cells to escape from growth arrest and to resume proliferation. This should happen even if the intervention into the feedback loop was started at a late time point (e.g. 6 days) after induction of senescence.
To analyse DNA damage foci dynamics we used a reporter construct (AcGFP–53BP1c) that quantitatively reports single DNA damage foci kinetics in time-resolved live cell microscopy (Nelson et al, 2009). Foci frequency measurements quantitatively confirmed the prediction from the stochastic model. More importantly, we found that many individual foci in both telomere- and stress-dependent senescence had short lifespans with half-lives below 15 h. Feedback loop inhibition reduced only the frequencies of short-lived DNA damage foci in accordance with the hypothesis that ROS production contributed to DDR by constant replenishment of short-lived DNA damage foci.
Finally, we inhibited signalling through the loop at different time points after induction of senescence by ionizing radiation and measured ROS levels, DNA damage foci frequencies and proliferation markers. Treatments with the MAPK14 inhibitor SB203580 or the free radical scavenger PBN were used to block the loop. The results quantitatively confirmed the model prediction and indicated that the feedback loop between DDR and ROS production was both necessary and sufficient to maintain cell cycle arrest for at least 6–10 days after induction of senescence. Interestingly, the loop was still active at later time points and in deep senescence, but proliferation arrest was then stabilized by additional factor(s). This indicated that certain features of the senescent phenotype-like ROS production that might be responsible for the negative impact of senescent cells into their tissue environment can be successfully inhibited even in deep senescence. This may prove relevant for novel therapeutic studies aiming to modulate intracellular ROS levels in both aging and cancer.
Cellular senescence—the permanent arrest of cycling in normally proliferating cells such as fibroblasts—contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of ‘deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFβ. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
PMCID: PMC2835567  PMID: 20160708
aging; cell senescence; DNA damage foci; mitochondria; reactive oxygen
16.  The A78V Mutation in the Mad3-like Domain of Schizosaccharomyces pombe Bub1p Perturbs Nuclear Accumulation and Kinetochore Targeting of Bub1p, Bub3p, and Mad3p and Spindle Assembly Checkpoint Function 
Molecular Biology of the Cell  2005;16(1):385-395.
During mitosis, the spindle assembly checkpoint (SAC) responds to faulty attachments between kinetochores and the mitotic spindle by imposing a metaphase arrest until the defect is corrected, thereby preventing chromosome missegregation. A genetic screen to isolate SAC mutants in fission yeast yielded point mutations in three fission yeast SAC genes: mad1, bub3, and bub1. The bub1-A78V mutant is of particular interest because it produces a wild-type amount of protein that is mutated in the conserved but uncharacterized Mad3-like region of Bub1p. Characterization of mutant cells demonstrates that the alanine at position 78 in the Mad3-like domain of Bub1p is required for: 1) cell cycle arrest induced by SAC activation; 2) kinetochore accumulation of Bub1p in checkpoint-activated cells; 3) recruitment of Bub3p and Mad3p, but not Mad1p, to kinetochores in checkpoint-activated cells; and 4) nuclear accumulation of Bub1p, Bub3p, and Mad3p, but not Mad1p, in cycling cells. Increased targeting of Bub1p-A78V to the nucleus by an exogenous nuclear localization signal does not significantly increase kinetochore localization or SAC function, but GFP fused to the isolated Bub1p Mad 3-like accumulates in the nucleus. These data indicate that Bub1p-A78V is defective in both nuclear accumulation and kinetochore targeting and that a threshold level of nuclear Bub1p is necessary for the nuclear accumulation of Bub3p and Mad3p.
PMCID: PMC539181  PMID: 15525673
17.  Chemical Genetics Reveals a Specific Requirement for Cdk2 Activity in the DNA Damage Response and Identifies Nbs1 as a Cdk2 Substrate in Human Cells 
PLoS Genetics  2012;8(8):e1002935.
The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1—a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling—dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR–sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.
Author Summary
Multiple cyclin-dependent kinases (CDKs) control human cell proliferation, but it remains unclear how functions of different CDKs are coordinated during unperturbed cell division or after dividing cells incur DNA damage. DNA lesions activate checkpoint signaling pathways to inhibit CDK activity, arrest the cell division cycle, and thus prevent loss of genetic information; but an effective response to damage also requires CDK activity to modify components of repair and checkpoint pathways. We took a chemical-genetic approach to ask if a specific CDK, Cdk2, played a specialized, non-redundant role in protecting genomic integrity of human cells. By sensitizing Cdk2 to chemical inhibition, we were able to detect a specific requirement for its catalytic activity in survival of cells after exposure to ionizing radiation (IR). We identified Nbs1, product of the gene mutated in the cancer-predisposing Nijmegen Breakage Syndrome, as a Cdk2 substrate and showed that mutant forms of Nbs1 that cannot be modified by Cdk2 are defective in protecting cells from death due to IR–induced DNA damage. Therefore, our work defines a DNA damage response pathway that depends on catalytic activity of a specific CDK in human cells and suggests a mechanism to promote efficient repair without triggering inappropriate cell division.
PMCID: PMC3426557  PMID: 22927831
18.  Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells 
Molecular Human Reproduction  2010;16(9):654-664.
Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex—(APC–CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC–CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic cells.
PMCID: PMC2930517  PMID: 20453035
resumption of meiosis; prophase I arrest; oocyte; G2-checkpoint; checkpoint recovery
19.  Cell cycle control, checkpoint mechanisms, and genotoxic stress. 
Environmental Health Perspectives  1999;107(Suppl 1):5-24.
The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling.
PMCID: PMC1566366  PMID: 10229703
20.  Mitotic Arrest-Associated Apoptosis Induced by Sodium Arsenite in A375 Melanoma Cells Is BUBR1-Dependent 
A375 human malignant melanoma cells undergo mitotic arrest-associated apoptosis when treated with pharmacological concentrations of sodium arsenite, a chemotherapeutic for acute promyelocytic leukemia. Our previous studies indicated that decreased arsenite sensitivity correlated with reduced mitotic spindle checkpoint function and reduced expression of the checkpoint protein BUBR1. In the current study, arsenite induced securin and cyclin B stabilization, BUBR1 phosphorylation, and spindle checkpoint activation. Arsenite also increased activating cyclin dependent kinase 1 (CDK1) Thr161 phosphorylation but decreased inhibitory Tyr15 phosphorylation. Mitotic arrest resulted in apoptosis as indicated by colocalization of mitotic phospho-Histone H3 with active caspase 3. Apoptosis was associated with BCL-2 Ser70 phosphorylation. Inhibition of CDK1 with roscovitine in arsenite-treated mitotic cells inhibited spindle checkpoint maintenance as inferred from reduced BUBR1 phosphorylation, reduced cyclin B expression, and diminution of mitotic index. Roscovitine also reduced BCL-2 Ser70 phosphorylation and protected against apoptosis, suggesting mitotic arrest caused by hyperactivation of CDK1 directly or indirectly leads to BCL-2 phosphorylation and apoptosis. In addition, suppression of BUBR1 with siRNA prevented arsenite-induced mitotic arrest and apoptosis. These findings provide insight into the mechanism of arsenic’s chemotherapeutic action and indicate a functional spindle checkpoint may be required for arsenic-sensitivity.
PMCID: PMC2806883  PMID: 18501396
Arsenite; mitotic arrest; apoptosis; spindle checkpoint
21.  p53 Prevents Immature Escaping from Cell Cycle G2 Checkpoint Arrest through Inhibiting cdk2-dependent NF-Y Phosphorylation 
Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and this function is required for the maintenance of genomic integrity. In this study, we addressed a role of p53 in escaping from cell cycle G2 arrest following DNA damage.
Materials and Methods
Cell cycle checkpoint arrest in the human colon cancer cell line HCT116 and its derivatives carry p53 or p21 deletions, were examined by FACS analysis, immunoprecipitation, Western blot and IP-kinase assay.
While the cells with functional p53 were arrested at both the G1 and G2 checkpoints, the p53-deficient cells failed to arrest at G1, but they were arrested at G2. However, the p53-deficient cells failed to sustain G2 checkpoint arrest and they entered mitosis earlier than did the p53-positive cells and so this resulted in extensive cell death. Cdc2 kinase becomes reactivated in p53-deficient cells in association with entry into mitosis, but not in the p53-positive cells. Upon DNA damage, the p21-deficient cells, like the p53-negative cells, not only failed to repress cdk2-dependent NF-Y phosphorylation, but they also failed to repress the expression of such cell cycle G2-regulatory genes as cdc2, cyclin B, RNR-R2 and cdc25C, which have all been previously reported as targets of NF-Y transcription factor.
p53 is essential to prevent immature escaping from cell cycle G2 checkpoint arrest through p21-mediated cdk2 inactivation, and this leads to inhibition of cdk2-dependent NF-Y phosphorylation and NF-Y dependent transcription of the cell cycle G2-rgulatory genes, including cdc2 and cyclin B.
PMCID: PMC2741649  PMID: 19771247
Tumor suppressor protein p53; Cell cycle; G2 phase; Cdc2 protein kinase; NF-Y protein
22.  Differential susceptibility of S and M phase cyclin/CDK complexes to inhibitory tyrosine phosphorylation in yeast 
Current biology : CB  2007;17(14):1181-1189.
Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B-type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation.
We demonstrate that in Saccharomyces cerevisiae several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27Kip1 similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved.
In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.
PMCID: PMC2034293  PMID: 17614281
CDK; Wee1; Cdc25; cyclin; SWE1; MIH1; CLB5; CLB2; CDC28
23.  Homeostatic control of polo-like kinase-1 engenders non-genetic heterogeneity in G2 checkpoint fidelity and timing 
Nature Communications  2014;5:4048.
The G2 checkpoint monitors DNA damage, preventing mitotic entry until the damage can be resolved. The mechanisms controlling checkpoint recovery are unclear. Here, we identify non-genetic heterogeneity in the fidelity and timing of damage-induced G2 checkpoint enforcement in individual cells from the same population. Single-cell fluorescence imaging reveals that individual damaged cells experience varying durations of G2 arrest, and recover with varying levels of remaining checkpoint signal or DNA damage. A gating mechanism dependent on polo-like kinase-1 (PLK1) activity underlies this heterogeneity. PLK1 activity continually accumulates from initial levels in G2-arrested cells, at a rate inversely correlated to checkpoint activation, until it reaches a threshold allowing mitotic entry regardless of remaining checkpoint signal or DNA damage. Thus, homeostatic control of PLK1 by the dynamic opposition between checkpoint signalling and pro-mitotic activities heterogeneously enforces the G2 checkpoint in each individual cell, with implications for cancer pathogenesis and therapy.
Cells exposed to DNA damage delay mitotic entry to allow repair. Liang et al. unexpectedly find that the duration of arrest and the completeness of repair vary from cell to cell, determined by progressively increasing polo-like kinase-1 activity, which must pass a threshold to trigger mitosis.
PMCID: PMC4059941  PMID: 24893992
24.  E2F4 Function in G2 
Cell cycle (Georgetown, Tex.)  2007;6(10):1147-1152.
Mammalian cells undergo cell cycle arrest in response to DNA damage through multiple checkpoint mechanisms. One such checkpoint pathway maintains genomic integrity by delaying mitotic progression in response to genotoxic stress. Transition though the G2 phase and entry into mitosis is considered to be regulated primarily by cyclin B1 and its associated catalytically active partner Cdk1. While not necessary for its initiation, the p130 and Rb-dependent target genes have emerged as being important for stable maintenance of a G2 arrest. It was recently demonstrated that by interacting with p130, E2F4 is present in the nuclei and plays a key role in the maintenance of this stable G2 arrest. Increased E2F4 levels and its translocation to the nucleus following genotoxic stress result in downregulation of many mitotic genes and as a result promote a G0-like state. Irradiation of E2F4-depleted cells leads to enhanced cellular DNA double-strand breaks that may be measured by comet assays. It also results in cell death that is characterized by caspase activation, sub-G1 and sub-G2 DNA content, and decreased clonogenic cell survival. Here we review these recent findings and discuss the mechanisms of G2 phase checkpoint activation and maintenance with a particular focus on E2F4.
PMCID: PMC2596058  PMID: 17507799
E2F4; p130; Rb; G2-phase; cell cycle; mitosis; ionizing radiation; genotoxic stress
25.  The Ulp2 SUMO Protease Is Required for Cell Division following Termination of the DNA Damage Checkpoint▿  
Molecular and Cellular Biology  2007;27(19):6948-6961.
Eukaryotic genome integrity is maintained via a DNA damage checkpoint that recognizes DNA damage and halts the cell cycle at metaphase, allowing time for repair. Checkpoint signaling is eventually terminated so that the cell cycle can resume. How cells restart cell division following checkpoint termination is poorly understood. Here we show that the SUMO protease Ulp2 is required for resumption of cell division following DNA damage-induced arrest in Saccharomyces cerevisiae, although it is not required for DNA double-strand break repair. The Rad53 branch of the checkpoint pathway generates a signal countered by Ulp2 activity following DNA damage. Interestingly, unlike previously characterized adaptation mutants, ulp2Δ mutants do not show persistent Rad53 phosphorylation following DNA damage, suggesting checkpoint signaling has been terminated and no longer asserts an arrest in these cells. Using Cdc14 localization as a cell cycle indicator, we show that nearly half of cells lacking Ulp2 can escape a checkpoint-induced metaphase arrest despite their inability to divide again. Moreover, half of permanently arrested ulp2Δ cells show evidence of an aberrant mitotic spindle, suggesting that Ulp2 is required for proper spindle dynamics during cell cycle resumption following a DNA damage-induced cell cycle arrest.
PMCID: PMC2099214  PMID: 17664284

Results 1-25 (1398040)