[Cell Cycle 3:11, 1383-1386; November 2004]; ©2004 Landes Bioscience
Checkpoint Adaptation and Recovery Extra Views
Back with Polo after the Break
*Correspondence to: René H. Medema; Division of Molecular Biology H8; The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital; Plesmanlaan121; Amsterdam 1066 CX The Netherlands; Tel: +31.0.20.5122096; Fax: +31.0.20. 6691383; Email:
[email protected] Received 09/18/04; Accepted 09/20/04
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1248
ACKNOWLEDGEMENTS
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In response to DNA damage or replication stress, checkpoint responses prevent further cell cycle progression to repair the damage or to delay the onset of mitosis until DNA replication is completed (reviewed in Zhou and Elledge).1 Besides enforcing a cell cycle arrest, checkpoint activation also actively promotes DNA repair and leads to upregulation of transcription. To accomplish this, a checkpoint signaling cascade exists consisting of DNA damage sensors, checkpoint-mediators and -effectors (reviewed by Melo and Toczyski).2 Depending on the type of damage, one of the two central players of the DNA damage response, ATM or ATR, is recruited to sites of damage to further activate effector kinases Chk1 and Chk2. In the G2 DNA damage checkpoint, both Chk1 and Chk2 inhibit cell cycle progression via activation of Wee1 on the one hand and inhibition of the Cdk-phosphatases Cdc25A, Cdc25B and Cdc25C on the other hand.3,4 The combination of these effects leads to a Y15-phosphorylated, inactive Cdk1 that is unable to trigger mitotic entry. Once a cell has arrested in G2 in response to DNA damage there are generally two options. If possible, a cell will repair the damaged DNA and reenter the cell cycle. During this so-called ‘recovery’ process, all of the damage is repaired, so that cells can divide without running the risk of transferring a damaged copy of the genome to their progeny. Alternatively, when the DNA damage cannot be repaired, a cell can undergo programmed cell death in order to eliminate the heavily damaged cells. This way, genomic integrity of the organism is warranted to a maximum extent. However, it was found that S. cerevisiae cells can eventually escape a sustained DNA damage-induced arrest and divide without repairing the damage.5 During this process, termed ‘adaptation’, the checkpoint signal that is generated from the damaged sites is gradually lost, allowing cyclin B-Cdk1 (Clb-Cdc28) activity to rise and drive mitotic entry.6 Genetic screens in S. cerevisiae have identified a number of genes that are required for checkpoint adaptation, including two subunits of casein kinase-II, the phosphatases Ptc2, Ptc3, the helicase Srs2 and the Polo kinase Cdc5.6-9 Mutant strains identified in these studies generally displayed normal cell cycle kinetics when unperturbed, but failed to escape a G2 arrest when unrepairable damage was present. The molecular mechanism underlying the requirement of Cdc5 in adaptation was not resolved, but the point-mutation (L251W) that was identified in the adaptationdefective mutant strain hinted at defective phosphorylation of (a subset of ) target proteins.8 More recent work by Pellicioli et al., showed that Cdc5 is required to inactivate checkpoint
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We appreciate the collaboration and/or discussions on angiogenesis and/or array technology with Judah Folkman, Lynn Hlatky, Phil Hahnfeldt (Harvard, Boston), Wilhelm Ansorge, Christian Schwager, Jonathon Blake (EMBL, Heidelberg), and Christian Maercker (RZPD). The authors would like to thank Dr. J. Haber for useful suggestions and critical reading of the manuscript. This work was supported by a grant from the Dutch Cancer Society (NKI 00-2191).
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cell cycle, genetic instability, cdc20 genotoxic stress
ce.
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KEY WORDS
S. cerevisiae cells that are unable to repair a double strand break ultimately escape the DNA damage checkpoint arrest and enter mitosis. This process called ‘adaptation’ depends on functional Cdc5, a Polo-like kinase, and was long thought to be limited to single-cell organisms. However, the recent finding that Xenopus extracts can adapt to a long-lasting stall in DNA replication indicates that checkpoint adaptation does also occur in multicellular organisms. Interestingly, the Xenopus Polo-like kinase (Plx1) plays an important role in this adaptation. To add to this, data from our laboratory have shown that the human Polo-like kinase (Plk1) is also required for cell cycle reentry following a DNA damage-induced arrest. But here, Plk1 was shown to be required for bona-fide checkpoint recovery, rather than adaptation. That is, Plk1 is required to restart the cell cycle once all of the damage is repaired and checkpoint signaling is turned off. While the target of Plx1 during adaptation is a component of the checkpoint machinery (Claspin), the target of Plk1 during recovery turns out to be a mitotic regulator (Wee1). Here, we discuss some of the remarkable similarities and subtle differences in the molecular mechanisms that control checkpoint adaptation and recovery, and the role of Polo-like kinases therein.
No tD ist r
Division of Molecular Biology; The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital; Amsterdam, The Netherlands
te
ABSTRACT
Marcel A.T.M. van Vugt René H. Medema
www.landesbioscience.com
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signaling, and suggested the presence of a feedback mechanism in which Cdc5 is inhibited by Rad53, while Cdc5 in turn controls inactivation of Rad53.6,10 Obviously, checkpoint adaptation does pose a serious threat to genomic integrity, and therefore it was long suspected that adaptation would not be allowed in multicellular organisms. In contrast, single cell organisms have little to lose when they attempt mitosis if DNA repair turns out to be ineffective. The damaged cell either proves to be genetically fit enough to contribute to population survival, or has sustained damage that will result in lethality or out-competition. This view must be changed however, as evidence for checkpoint adaptation in multicellular organisms was recently presented by Yoo et al.11 After a prolonged stall in DNA replication in Xenopus extracts, checkpoint signaling ultimately ceased so that cyclin B-Cdk1 activity increased to mediate nuclear envelope breakdown. Polα remained associated with stalled replication forks during adaptation, indicating that indeed checkpoint adaptation, not recovery, was responsible for the observed mitotic entry.11 Interestingly, the Cdc5 homologue Plx1 was found to regulate checkpoint adaptation in Xenopus, through phosphorylation of the checkpoint mediator Claspin.11 Plx1-mediated phosphorylation of Claspin resulted in displacement of Claspin from chromatin thereby blocking any further activation of Xchk1. Consequently, checkpoint signaling is quenched and mitotic entry is allowed.11,12 Thus, Plx1 can inactivate checkpoint signaling to promote mitotic entry in Xenopus extracts. This mechanism appears similar to that controlling checkpoint adaptation in S. cerevisiae, in which Cdc5 is required for efficient inactivation of the checkpoint kinase Rad53 (the budding yeast homologue of human Chk2).6 However, whether checkpoint adaptation serves a similar purpose in both species is unclear. In S. cerevisiae, checkpoint adaptation allows multiple subsequent cell divisions, indicating that in this organism adaptation truly is an attempt at survival if damage cannot be repaired.5 It is uncertain, however, what the faith of Xenopus cells is when they adapt to a permanent halt in replication. As the experiments were done with Xenopus extracts it is not feasible to examine the possible outcome of adaptation (i.e., apoptosis or production of proliferation-competent progeny). Therefore it is very well possible that adaptation in multicellular organisms is a means to drive cells that can not be repaired into a mitotic catastrophe, as suggested by Yoo et al.11 Hence, adaptation in multicellular organisms could facilitate efficient removal of cells with excess DNA damage (reviewed by Castedo et al).13 Indeed, recent evidence indicates that mitotic entry in the presence of DNA damage can lead to cell death.14 Therefore, it would be interesting to test if checkpoint adaptation occurs in intact Xenopus cells and determine whether cells can actually produce viable progeny. Also, it would be worthwhile to investigate whether checkpoint adaptation takes place in other multicellular organisms. In this respect, it is of interest to note that the serine-residue (S904) in Xenopus Claspin that is phosphorylated by Xatr to create a binding site for Plx1, is conserved in human Claspin.11 However, whereas S904 is located in an ATM/ATR consensus-phosphorylation motif in Xenopus Claspin, in human Claspin the corresponding serine lies within a Cdk1-consensus motif. Thus, if Plk1/Claspin-mediated checkpoint adaptation is conserved in human cells, the responsible mechanisms might be somewhat different. In conclusion, the fact that a Polo-like kinase regulates checkpoint adaptation in multicellular organisms is exciting and warrants further investigation to determine its exact outcome and impact on genomic integrity. 1384
While these data show that adaptation may also be an alternative means to deal with genotoxic stress in multicellular organisms, checkpoint recovery with fully repaired DNA must, without a doubt, be the most desired outcome after a checkpoint arrest. However, most of what we currently know about this process comes from work in yeast (for a review see ref.13), and we know very little about the recovery process in higher organisms. Therefore, we recently set out to investigate the requirements for checkpoint recovery in mammalian cells. To this end, checkpoint-arrested G2 cells were treated with caffeine to quench checkpoint signaling. Using this protocol, a cell cycle restart after DNA repair was mimicked, resembling checkpoint recovery. Surprisingly, whereas control cells rapidly entered mitosis after checkpoint silencing, Plk1-depleted cells did not.16 In addition, we found that the Cdk-phosphatase Cdc25B is equally important for a cell cycle restart following a DNA damage-induced arrest. In contrast, nondamaged cells did not require Plk1 or Cdc25B for mitotic entry. Importantly, we could demonstrate that the same dependence on Plk1 applies to cells that undergo true recovery, i.e., where repair is allowed to reach completion. Moreover, we found that Plk1 promotes the degradation of Wee1, a Cdk1 inhibitory kinase, during recovery. In fact, the requirement for Plk1 during recovery was completely lost in Wee1-depleted cells, indicating that this is the crucial target of Plk1 in this process. These data warrant an interesting new view on the roles of Plk1 and Wee1 during mitotic entry. Polo-like kinases have long been known to promote mitotic entry through direct phosphorylation of Cdc25C and cyclin B, whereas Wee1 controls inactivation of the cyclin B-Cdk1 complex.17-21 However, the exact contribution of Plk1 and Wee1 during mitotic entry and the cellular response to DNA damage has been a matter of strong debate. Our experiments now show that the cell cycle machinery functions fundamentally different in damaged versus nondamaged cells, and demonstrate that in mammalian cells the requirement for Plk1 and Wee1-degradation is restricted to cells recovering from a DNA damage-induced arrest.16 In S. cerevisiae and S. pombe, two model systems for studying DNA damage responses, several mutants were identified that specifically interfere with checkpoint recovery. One of the genes found to be essential for recovery is the S. pombe Slp1+ gene.22 Slp+ is the fission yeast homologue of Cdc20, an activator of the Anaphase-Promoting Complex/Cyclosome (APC/C) that controls degradation of mitotic cyclins and several other important mitotic regulators.23 Interestingly, the requirement for Slp1+ in checkpoint recovery is lost in cells lacking Wee1, similar to what was observed with the requirement for Plk1 in recovery.22 Whether this requirement for Slp+ reflects a conserved role for Cdc20 family members in DNA damage checkpoint recovery remains unclear, but it is interesting to note that Cdc20 is phosphorylated by Chk1 in response to DNA damage in S. cerevisiae.24 Even more so, in avian cells, Cdc20 is ubiquitinated and degraded in response to DNA damage and interference with Cdc20-ubiquitination results in a failure to maintain a G2 DNA damage arrest.25 Whether mammalian Cdc20 (p55cdc) is also required for checkpoint recovery remains to be elucidated, but we do find that Cdc20 protein levels drop in response to DNA damage and are quickly restored to normal levels during checkpoint recovery, coincident with mitotic entry (Fig. 1A). Why exactly Cdc20 needs to be degraded during a checkpoint arrest is unclear, but it could be required to prevent degradation of the mitotic cyclins during this arrest so that sufficiently high levels of cyclin A and cyclin B are around to promote an eventual restart of the cell cycle. In addition, as the presence of cyclin B also serves to prevent
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the type of arrest studied (i.e., stalled replication vs. DNA damage-induced arrest). Nonetheless, several adaptation-defective mutants identified in yeast are also impaired in checkpoint recovery, indicating that there is a certain degree of overlap in the regulatory proteins involved.7 However, a subset of adaptation-defective mutants, most notably the Cdc5ad mutant, can recover from DNA damage, suggesting that the two pathways are not completely identical.7 Of course one crucial difference remains the persistence of DNA or replication damage during adaptation, certainly demanding active silencing of the checkpoint. But if checkpoint recovery also requires active silencing or occurs via a passive demise of the checkpoint signal once the source of the signal is taken away, is unclear. Experiments done in S. pombe have shown that checkpoint recovery involves Cdc2-mediated phosphorylation of the checkpoint component Crb2, suggesting that it is an active process.27 However, it has not been resolved if this phosphorylation has an impact on repair of the damage, or plays a role in active quenching of the checkpoint. Most of our studies done on checkpoint recovery in human cells involved checkpointinhibition by caffeine and therefore active mechanisms for checkpoint silencing were no longer rate limiting under these conditions.16 Thus, whereas our studies demonstrated that Plk1 is essential to actively promote mitotic entry during recovery, they Figure 1. (A) Cdc20 is degraded after DNA damage but recovers during checkpoint recovery. U2OS did not rule out a role for Plk1 in the active cells were treated with thymidine for 24 h to block cells at the G1-S transition. After release from the silencing of the checkpoint during recovery thymidine block, cells were treated with 0.25 µM doxorubicin to activate the G2 DNA damage as well. Moreover, although the nature of the checkpoint. Judged by FACS analysis, all cells were arrested at the G2-M transition after doxorubicin treatment. Subsequently, checkpoint signaling was silenced by caffeine (5 mM) addition. Cells were primary Polo-like kinase target appears to be harvested at indicated time-points and immunoblotted for Cdk4 (rabbit anti-Cdk4, Santa-Cruz different in each case (the cell cycle regulator Biotechnology) and Cdc20 (goat anti-Cdc20, Santa Cruz Biotechnology). (B) Checkpoint recovery Wee1 during checkpoint recovery in mamversus checkpoint adaptation. The mechanisms that control checkpoint adaptation and checkpoint malian cells opposed to the checkpoint recovery in different species are schematically depicted (Black arrows represent checkpoint signaling, adaptor Claspin during Xenopus adaptation), grey arrows represent recovery/adaptation signaling). The left box represents checkpoint adaptation based on the available data one cannot in S. cerevisiae and X. leavis. The right box represents checkpoint recovery in S. pombe and H. sapiens. All depicted species detect DNA damage through P(I)3-kinase-related DNA damage sensors, notably exclude that Plk1 acts to phosphorylate both Mec1, ATR, Rad3 and ATM. Subsequently, activated downstream checkpoint kinases (Rad53, Chk1, during recovery. Homologues of Claspin Chk2, with adaptor proteins Mrc1 and Claspin) mediate inhibition of Cdc25 phosphatases to inhibit have been identified in S. cerevisiae (Mrc1) Cyclin B-Cdk1 activity. During checkpoint adaptation, Polo-like kinases Cdc5 and Plx1 are required in and H. Sapiens (hClaspin), but the roles of S. cerevisiae and Xenopus respectively. Also, in budding yeast, Ptc2 and Ptc3 are essential for adaptation. these Claspin homologues in checkpoint Wee1 homologues are degraded during mitotic entry in S. cerevisiae and Xenopus, a process that recovery has not been addressed to date requires Cdc5 in S. cerevisiae. Whether Wee1 degradation is involved in adaptation, however, is (Fig. 1).12,28,29 Nonetheless, the notion that unknown. Recovery in human cells requires Plk1-dependent Wee1 degradation. Whether Plk1 or phosphatases are required for checkpoint- inactivation is not known. In fission yeast, however, Dis1 the Cdc5 adaptation-defective mutant can recover is puzzling in this respect, as it phosphatase-activity was shown to be required for checkpoint recovery (see text for further details). suggests that Cdc5 is not required for unscheduled replication, degradation of Cdc20 during a DNA dam- checkpoint recovery in S. cerevisiae.7 This discrepancy may be due age-induced arrest could be required to prevent DNA rereplication.26 to a difference in the amount of damage induced in the two experiOne important issue that arises from the observations made thus mental systems. Alternatively, as the Cdc5 adaptation-defective far is whether checkpoint adaptation and recovery are related mutant is not a complete null mutant, it is possible that its function processes that use (partially) overlapping mechanisms (Fig. 1B). It is in promoting mitotic entry (i.e., phosphorylation of Wee1) is important to realize when comparing different studies, that the retained, while its role in adaptation (i.e., inactivation of Rad53) is mechanism underling adaptation/recovery may alter depending on impaired. To address this issue we will need to analyze if a corresponding www.landesbioscience.com
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mutation in Plk1 affects its function in recovery in mammalian cells. An additional hint that recovery as well as adaptation does require active checkpoint silencing, is provided by the observation that both processes depend on phosphatase activity in yeast cells (Fig. 1). The phosphatase Dis2 was found to be essential for checkpoint recovery after double strand breaks in S. pombe and acts to inactivate Chk1 through direct dephosphorylation.30 Likewise, the Ptc2 and Ptc3 phosphatases were shown to be essential for both adaptation and recovery in S. cerevisiae through Rad53 inactivation.9 However, whether these phosphatases act constitutively, or require specific activation during adaptation or recovery and whether similar phosphatases regulate human checkpoint recovery or adaptation is unclear. Another common feature that proposes a relation between checkpoint recovery and adaptation concerns the requirement for Wee1-degradation (Fig. 1B). In human cells, degradation of Wee1 through phosphorylation by Plk1 controls checkpoint recovery.16 Moreover, mitotic entry after a stalled replication in Xenopus extracts also requires destruction of Wee1, but if this requires direct phosphorylation by Plx1 remains to be proven.31 Such a requirement for Polo-like kinases in Wee1-degradation was found in S. cerevisiae, but the impact of this pathway on recovery or adaptation in budding yeast has not been evaluated.32 Taken together, these notions strongly suggest that checkpoint recovery and checkpoint adaptation occur through evolutionary conserved pathways that (at least) partially overlap. One would suspect that whereas adaptation may allow further proliferation in single cell organisms, it should be tightly coupled to programmed cell death in multicellular organisms, otherwise it poses a serious threat to the genomic integrity of the organism. Thus, further research is warranted to evaluate the eventual outcome of adaptation in multicellular organisms. In addition, it will be interesting to know if checkpoint adaptation also occurs in human cells, and if there is a role for Plk1 in this process. Finally, the mechanism of checkpoint silencing during recovery (i.e., active or inactive) will need to be addressed. These studies will not only help us understand better how mammalian cells can deal with genotoxic stress, but also if there is any merit in modulating Plk1 or Cdc25B activity as adjuvant therapy in combination with genotoxic drugs. References 1. Zhou BB, Elledge SJ. The DNA damage response: Putting checkpoints in perspective. Nature 2000; 408:433-9. 2. Melo J, Toczyski D. A unified view of the DNA-damage checkpoint. Curr Opin Cell Biol 2002; 14:237-45. 3. Donzelli M, Draetta GF. Regulating mammalian checkpoints through Cdc25 inactivation. EMBO Rep 2003; 4:671-7. 4. O’Connell MJ, Raleigh JM, Verkade HM, Nurse P. Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. Embo J 1997; 16:545-54. 5. Sandell LL, Zakian VA. Loss of a yeast telomere: Arrest, recovery, and chromosome loss. Cell 1993; 75:729-39. 6. Pellicioli A, Lee SE, Lucca C, Foiani M, Haber JE. Regulation of saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. Mol Cell 2001; 7:293-300. 7. Vaze MB, Pellicioli A, Lee SE, Ira G, Liberi G, Arbel-Eden A, Foiani M, Haber JE. Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol Cell 2002; 10:373-85. 8. Toczyski DP, Galgoczy DJ, Hartwell LH. CDC5 and CKII control adaptation to the yeast DNA damage checkpoint. Cell 1997; 90:1097-106. 9. Leroy C, Lee SE, Vaze MB, Ochsenbien F, Guerois R, Haber JE, Marsolier-Kergoat MC. PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break. Mol Cell 2003; 11:827-35. 10. Sanchez Y, Bachant J, Wang H, Hu F, Liu D, Tetzlaff M, Elledge SJ. Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 1999; 286:1166-71. 11. Yoo HY, Kumagai A, Shevchenko A, Dunphy WG. Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase. Cell 2004; 117:575-88.
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12. Kumagai A, Dunphy WG. Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol Cell 2000; 6:839-49. 13. Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G. Cell death by mitotic catastrophe: A molecular definition. Oncogene 2004; 23:2825-37. 14. Nitta M, Kobayashi O, Honda S, Hirota T, Kuninaka S, Marumoto T, Ushio Y, Saya H. Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents. Oncogene 2004; 23:6548-58. 15. Lee SE, Pellicioli A, Demeter J, Vaze MP, Gasch AP, Malkova A, Brown PO, Botstein D, Stearns T, Foiani M, Haber JE. Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol 2000; 65:303-14. 16. Van Vugt MA, Bras A, Medema RH. Polo-like kinase-1 controls recovery from a DNA damage-induced arrest in mammalian cells. Mol Cell 2004; 15:1-20. 17. Yuan J, Eckerdt F, Bereiter-Hahn J, Kurunci-Csacsko E, Kaufmann M, Strebhardt K. Cooperative phosphorylation including the activity of polo-like kinase 1 regulates the subcellular localization of cyclin B1. Oncogene 2002; 21:8282-92. 18. McGowan CH, Russell P. Human wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J 1993; 12:75-85. 19. Parker LL, Piwnica-Worms H. Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science 1992; 257:1955-7. 20. Qian YW, Erikson E, Taieb FE, Maller JL. The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes. Mol Biol Cell 2001; 12:1791-9. 21. Jackman M, Lindon C, Nigg EA, Pines J. Active cyclin B1-Cdk1 first appears on centrosomes in prophase. Nat Cell Biol 2003; 5:143-8. 22. Matsumoto T. A fission yeast homolog of CDC20/p55CDC/Fizzy is required for recovery from DNA damage and genetically interacts with p34cdc2. Mol Cell Biol 1997; 17:742-50. 23. Hwang LH, Lau LF, Smith DL, Mistrot CA, Hardwick KG, Hwang ES, Amon A, Murray AW. Budding yeast Cdc20: A target of the spindle checkpoint. Science 1998; 279:1041-4. 24. Searle JS, Schollaert KL, Wilkins BJ, Sanchez Y. The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression. Nat Cell Biol 2004; 6:138-45. 25. Sudo T, Ota Y, Kotani S, Nakao M, Takami Y, Takeda S, Saya H. Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells. EMBO J 2001; 20:6499-508. 26. Wuarin J, Buck V, Nurse P, Millar JB. Stable association of mitotic cyclin B/Cdc2 to replication origins prevents endoreduplication. Cell 2002; 111:419-31. 27. Esashi F, Yanagida M. Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint. Mol Cell 1999; 4:167-74. 28. Tanaka K, Russell P. Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1. Nat Cell Biol 2001; 3:966-72. 29. Alcasabas AA, Osborn AJ, Bachant J, Hu F, Werler PJ, Bousset K, Furuya K, Diffley JF, Carr AM, Elledge SJ. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat Cell Biol 2001; 3:958-65. 30. Den Elzen NR, O’Connell MJ. Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1. EMBO J 2004; 23:908-18. 31. Michael WM, Newport J. Coupling of mitosis to the completion of S phase through Cdc34-mediated degradation of Wee1. Science 1998; 282:1886-9. 32. Sakchaisri K, Asano S, Yu LR, Shulewitz MJ, Park CJ, Park JE, Cho YW, Veenstra TD, Thorner J, Lee KS. Coupling morphogenesis to mitotic entry. Proc Natl Acad Sci USA 2004; 101:4124-9.
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