Plx1 is required for chromosomal DNA replication under stressful conditions

Polo-like kinase (Plk)1 is required for mitosis progression. However, although Plk1 is expressed throughout the cell cycle, its function during S-phase is unknown. Using Xenopus laevis egg extracts, we demonstrate that Plx1, the Xenopus orthologue of Plk1, is required for DNA replication in the presence of stalled replication forks induced by aphidicolin, etoposide or reduced levels of DNA-bound Mcm complexes. Plx1 binds to chromatin and suppresses the ATM/ATR-dependent intra-S-phase checkpoint that inhibits origin firing. This allows Cdc45 loading and derepression of DNA replication initiation. Checkpoint activation increases Plx1 binding to the Mcm complex through its Polo box domain. Plx1 recruitment to chromatin is independent of checkpoint mediators Tipin and Claspin. Instead, ATR-dependent phosphorylation of serine 92 of Mcm2 is required for the recruitment of Plx1 to chromatin and for the recovery of DNA replication under stress. Depletion of Plx1 leads to accumulation of chromosomal breakage that is prevented by the addition of recombinant Plx1. These data suggest that Plx1 promotes genome stability by regulating DNA replication under stressful conditions.     

A novel role for Greatwall kinase in recovery from DNA damage

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression. The shut off process of the DDR upon satisfaction of DNA repair, also known as “checkpoint recovery,” involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G₂/M transition and helps maintain M phase through inhibition of PP2A/B55δ, the principal phosphatase for Cdk-phosphorylated substrates. Here, we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild-type, but not kinase-dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl overexpression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA damage.Key words: Greatwall,; DNA damage; checkpoint recovery     

A novel role for Greatwall kinase in recovery from DNA damage

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression (Halazonetis et al., 2008; Motoyama and Naka, 2004; Zhou and Elledge, 2000). The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G2/M transition (Archambault et al., 2007; Jackson, 2006; Zhao et al., 2008; Yu et al., 2004; Yu et al., 2006; Zhao et al., 2006) and helps maintain M phase through inhibition of PP2A/B55δ (Burgess et al., 2010; Castilho et al., 2009; Goldberg, 2010; Lorca et al., 2010; Vigneron et al., 2009), the principal phosphatase for Cdk-phosphorylated substrates. Here we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild type, but not kinase dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl over-expression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA     

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.     

Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase

The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. We show that, during this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response.     

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.     

Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores

Dynamic attachment of microtubules to kinetochores during mitosis generates pulling force, or tension, required for the high fidelity of chromosome separation. A lack of tension activates the spindle checkpoint and delays the anaphase onset. A key step in the tension-response pathway involves the phosphorylation of the 3F3/2 epitope by an unknown kinase on untensed kinetochores. Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation. Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores. Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores. Thus, Plx1 couples the tension signal to cellular responses through phosphorylating the 3F3/2 epitope and targeting structural and checkpoint proteins to kinetochores.     

Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

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.Key words: DNA damage checkpoint, Ras signaling, budding yeast, cAMP-dependent protein kinase, anaphase promoting complex, neurofibromatosis type 1     

Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

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 ira1D ira2D 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.     

Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.     

The Polo-like kinase Plx1 interacts with and inhibits Myt1 after fertilization of Xenopus eggs

During the meiotic cell cycle in Xenopus oocytes, p90(rsk), the downstream kinase of the Mos-MAPK pathway, interacts with and inhibits the Cdc2 inhibitory kinase Myt1. However, p90(rsk) is inactivated after fertilization due to the degradation of Mos. Here we show that the Polo-like kinase Plx1, instead of p90(rsk), interacts with and inhibits Myt1 after fertilization of Xenopus eggs. At the M phase of the embryonic cell cycle, Cdc2 phosphorylates Myt1 on Thr478 and thereby creates a docking site for Plx1. Plx1 can phosphorylate Myt1 and inhibit its kinase activity both in vitro and in vivo. The interaction between Myt1 and Plx1 is required, at least in part, for normal embryonic cell divisions. Finally, and interestingly, Myt1 is phosphorylated on Thr478 even during the meiotic cell cycle, but its interaction with Plx1 is largely inhibited by p90(rsk)-mediated phosphorylation. These results indicate a switchover in the Myt1 inhibition mechanism at fertilization of Xenopus eggs, and strongly suggest that Plx1 acts as a direct inhibitory kinase of Myt1 in the mitotic cell cycles in Xenopus.     

Regulation of Op18 during spindle assembly in Xenopus egg extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.     

Protein phosphatase 1 is essential for Greatwall inactivation at mitotic exit

Entry into mitosis is mediated by the phosphorylation of key cell cycle regulators by cyclin-dependent kinase 1 (Cdk1). In Xenopus embryos, the M-phase-promoting activity of Cdk1 is antagonized by protein phosphatase PP2A-B55. Hence, to ensure robust cell cycle transitions, Cdk1 and PP2A-B55 must be regulated so that their activities are mutually exclusive. The mechanism underlying PP2A-B55 inactivation at mitotic entry is well understood: Cdk1-activated Greatwall (Gwl) kinase phosphorylates Ensa/Arpp19, thereby enabling them to bind to and inhibit PP2A-B55. However, the re-activation of PP2A-B55 during mitotic exit, which is essential for cell cycle progression, is less well understood. Here, we identify protein phosphatase PP1 as an essential component of the PP2A-B55 re-activation pathway in Xenopus embryo extracts. PP1 initiates the re-activation of PP2A-B55 by dephosphorylating Gwl. We provide evidence that PP1 targets the auto-phosphorylation site of Gwl, resulting in efficient Gwl inactivation. This step is necessary to facilitate subsequent complete dephosphorylation of Gwl by PP2A-B55. Thus, by identifying PP1 as the phosphatase initiating Gwl inactivation, our study provides the molecular explanation for how Cdk1 inactivation is coupled to PP2A-B55 re-activation at mitotic exit.     

Loading of the 3F3/2 antigen onto kinetochores is dependent on the ordered assembly of the spindle checkpoint proteins

Accurate chromosome segregation is controlled by the spindle checkpoint, which responds to the lack of microtubule-kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B, and Plk1/Plx1 kinases. The presence of the 3F3/2 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1 but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopus extracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, and BubR1 and Plx1 second, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid in the eventual identification of the 3F3/2 substrate.     

Cdk1 phosphorylation of BubR1 controls spindle checkpoint arrest and Plk1-mediated formation of the 3F3/2 epitope

Accurate chromosome segregation is controlled by the spindle checkpoint, which senses kinetochore– microtubule attachments and tension across sister kinetochores. An important step in the tension-signaling pathway involves the phosphorylation of an unknown protein by polo-like kinase 1/Xenopus laevis polo-like kinase 1 (Plx1) on kinetochores lacking tension to generate the 3F3/2 phosphoepitope. We report here that the checkpoint protein BubR1 interacts with Plx1 and that phosphorylation of BubR1 by Plx1 generates the 3F3/2 epitope. Formation of the BubR1 3F3/2 epitope by Plx1 requires a prior phosphorylation of BubR1 on Thr 605 by cyclin-dependant kinase 1 (Cdk1). This priming phosphorylation of BubR1 by Cdk1 is required for checkpoint-mediated mitotic arrest and for recruitment of Plx1 and the checkpoint protein Mad2 to unattached kinetochores. Biochemically, formation of the 3F3/2 phosphoepitope by Cdk1 and Plx1 greatly enhances the kinase activity of BubR1. Thus, Cdk1-mediated phosphorylation of BubR1 controls checkpoint arrest and promotes the formation of the kinetochore 3F3/2 epitope.     

A feedback loop in the polo-like kinase activation pathway

The Xenopus polo-like kinase Plx1 plays important roles during entry into and exit from mitosis (M phase). Previous studies revealed that Plx1 is activated by phosphorylation on serine and threonine residues, and purification of an activating enzyme from mitotic Xenopus egg extracts led to cloning and characterization of Xenopus polo-like kinase kinase (xPlkk1), which can phosphorylate and activate Plx1 in vitro. In the present study, a positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other. Sequencing of radiolabeled xPlkk1 after phosphorylation by Plx1 in vitro identified three phosphorylation sites each spaced three amino acids apart, two of which have the consensus acidic-X-pSer-hydrophobic described for other polo-like kinase substrates. In addition, endogenous xPlkk1 in oocytes was phosphorylated on these sites in M phase but not in interphase. A mutant xPlkk1 in which these three amino acids were changed to alanine (xPlkk1(SA3)) was unable to be phosphorylated or activated in vitro by Plxl. Depletion of Plx1 from oocyte extracts prior to stimulation of the G(2)/M transition blocked the activation of xPlkk1, but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G(2)/M transition.     

TopBP1 activates the ATR-ATRIP complex

ATR is a key regulator of checkpoint responses to incompletely replicated and damaged DNA, but the mechanisms underlying control of its kinase activity are unknown. TopBP1, the vertebrate homolog of yeast Cut5/Dbp11, has dual roles in initiation of DNA replication and regulation of checkpoint responses. We show that recombinant TopBP1 induces a large increase in the kinase activity of both Xenopus and human ATR. The ATR-activating domain resides in a conserved segment of TopBP1 that is distinct from its numerous BRCT repeats. The isolated ATR-activating domain from TopBP1 induces ectopic activation of ATR-dependent signaling in both Xenopus egg extracts and human cells. Furthermore, Xenopus egg extracts containing a version of TopBP1 with an inactivating point mutation in the ATR-activating domain are defective in checkpoint regulation. These studies establish that activation of ATR by TopBP1 is a crucial step in the initiation of ATR-dependent signaling processes.     

Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts

We have identified Claspin, a novel protein that binds to Xenopus Chk1 (Xchk1). Binding of Claspin to Xchk1 is highly elevated in the presence of DNA templates that trigger a checkpoint arrest of the cell cycle in Xenopus egg extracts. Xchk1 becomes phosphorylated during a checkpoint response, and we demonstrate directly that this phosphorylation results in the activation of Xchk1. Immunodepletion of Claspin from egg extracts abolishes both the phosphorylation and activation of Xchk1. Furthermore, Claspin-depleted extracts are unable to arrest the cell cycle in response to DNA replication blocks. Taken together, these findings indicate that Claspin is an essential upstream regulator of Xchk1.     

Condensin recruitment to chromatin is inhibited by Chk2 kinase in response to DNA damage

The DNA damage checkpoint, when activated in response to genotoxic damage during S phase, arrests cells in G2 phase of the cell cycle. ATM, ATR, Chk1 and Chk2 kinases are the main effectors of this checkpoint pathway. The checkpoint kinases prevent the onset of mitosis by eliciting well characterized inhibitory phosphorylation of Cdk1. Since Cdk1 is required for the recruitment of condensin, it is thought that upon DNA damage the checkpoint also indirectly blocks chromosome condensation via Cdk1 inhibition. Here we report that the G2 damage checkpoint prevents stable recruitment of the chromosome-packaging-machinery components condensin complex I and II onto the chromatin even in the presence of an active Cdk1. DNA damage-induced inhibition of condensin subunit recruitment is mediated specifically by the Chk2 kinase, implying that the condensin complexes are targeted by the checkpoint in response to DNA damage, independently of Cdk1 inactivation. Thus, the G2 checkpoint directly prevents stable recruitment of condensin complexes to actively prevent chromosome compaction during G2 arrest, presumably to ensure efficient repair of the genomic damage.     

Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses

In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. In this study, we describe the isolation of a cDNA encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, we have identified the essential replication protein Mcm2 as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 underwent phosphorylation at Ser(92) in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involved both ATM and ATR, but the relative contribution of each kinase depended upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylated Mcm2 directly at Ser(92) in cell-free kinase assays. Immunodepletion of both ATM and ATR abrogated the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts.