The DNA Damage Checkpoint Signal in Budding Yeast Is Nuclear Limited (original) (raw)
The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage
Proceedings of the National Academy of Sciences, 2007
A single HO endonuclease-induced double-strand break (DSB) is sufficient to activate the DNA damage checkpoint and cause Saccharomyces cells to arrest at G 2 /M for 12–14 h, after which cells adapt to the presence of the DSB and resume cell cycle progression. The checkpoint signal leading to G 2 /M arrest was previously shown to be nuclear-limited. Cells lacking ATR-like Mec1 exhibit no DSB-induced cell cycle delay; however, cells lacking Mec1's downstream protein kinase targets, Rad53 or Chk1, still have substantial G 2 /M delay, as do cells lacking securin, Pds1. This delay is eliminated only in the triple mutant chk1 Δ rad53 Δ pds1 Δ, suggesting that Rad53 and Chk1 control targets other than the stability of securin in enforcing checkpoint-mediated cell cycle arrest. The G 2 /M arrest in rad53 Δ and chk1 Δ revealed a unique cytoplasmic phenotype in which there are frequent dynein-dependent excursions of the nucleus through the bud neck, without entering anaphase. Such excursi...
DNA damage checkpoint execution and the rules of its disengagement
Frontiers in Cell and Developmental Biology
Chromosomes are susceptible to damage during their duplication and segregation or when exposed to genotoxic stresses. Left uncorrected, these lesions can result in genomic instability, leading to cells’ diminished fitness, unbridled proliferation or death. To prevent such fates, checkpoint controls transiently halt cell cycle progression to allow time for the implementation of corrective measures. Prominent among these is the DNA damage checkpoint which operates at G2/M transition to ensure that cells with damaged chromosomes do not enter the mitotic phase. The execution and maintenance of cell cycle arrest are essential aspects of G2/M checkpoint and have been studied in detail. Equally critical is cells’ ability to switch-off the checkpoint controls after a successful completion of corrective actions and to recommence cell cycle progression. Interestingly, when corrective measures fail, cells can mount an unusual cellular response, termed adaptation, where they escape checkpoint a...
Molecular Cell, 2000
A regulatory network of proteins has been identified that participates in DNA damage checkpoint signaling. Central to this network is the ATM protein, the product of the gene mutated in the human disease ataxia-telangiectasia Savitsky et al., 1995). Ataxia-telangiectasia (A-T) Development is an autosomal recessive disease that displays a com- † Department of Dermatology plex phenotype (Boder and Sedgwick, 1970; Shiloh, ‡ Institute of Cancer Research 1998). Patients exhibit a progressive cerebellar ataxia, Columbia University in addition to severe immune deficiencies, gonadal atro-New York, NY 10032 phy, telangiectases, increased risk for cancer-partic- § Dipartimento di Biologia e Patologia Molecolare ularly lymphomas-and radiation sensitivity. Cell lines e Cellulare "L. Califano" from A-T patients show enhanced radiosensitivity to II Medical School ionizing radiation (Lavin and Shiloh, 1997), increased University of Naples chromosomal loss and breakage, and abnormal telo-80131 Naples mere morphology (Smilenov et al., 1997; Vaziri et al., Italy 1997). A-T cells are defective in cell cycle checkpoints Dipartimento di Medicina Sperimentale "G. Salvatore" in G1, S, and G2 (Meyn, 1995; Beamish et al., 1996; Hoekstra, 1997). The cellular phenotype of A-T suggests Medical School a defect in handling DNA breaks formed by external University of Catanzaro insults or as a result of normal physiological processes 88100 Catanzaro such as meiotic recombination and maturation of the Italy immune system. ATM is a serine/threonine kinase for which structurally and functionally similar proteins have been identified and characterized in S. cerevisiae, S. Summary pombe, D. melanogaster, and X. laevis (Weinert, 1992; Hari et al., 1995; Bentley et al., 1996; Robertson et al., Cell cycle checkpoints lead to the inhibition of cell 1999; Sibon et al., 1999). When exposed to ionizing radicycle progression following DNA damage. A cell-free ation, mammalian ATM -/cells cannot prevent S phase system derived from Xenopus eggs has been estabentry and undergo radio-resistant DNA synthesis (RDS) lished that reconstitutes the checkpoint pathway in-(Jeggo et al., 1998). Following ionizing radiation, ATM phosphorylates and hibiting DNA replication initiation. DNA containing participates in the activation of p53 (Banin et al., 1998; double-strand breaks inhibits replication initiation in a Canman et al., 1998; Khanna et al., 1998). Activated dose-dependent manner. Upon checkpoint activation, p53 promotes the synthesis of p21, a cyclin-dependent a prereplicative complex is assembled that contains kinase inhibitor with preferential affinity for Cdk2/ ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. CyclinE, delaying cell cycle prior to S phase entry. ATM The checkpoint is ATM dependent. Cdk2/CyclinE acts also phosphorylates and activates the Chk1 and Chk2 downstream of ATM and is downregulated by Cdk2 protein kinases (Matsuoka et al., 1998; Chaturvedi et phosphorylation on tyrosine 15. Cdk2AF/CyclinE is real., 1999; Chen et al., 1999). The activation of Chk1 is fractory to checkpoint signaling, and Cdc25A overessential to prevent entry into mitosis in mammalian rides the checkpoint and restores DNA replication. cells and in Xenopus extracts following DNA replication This report provides the description of a DNA damage block (Kumagai et al., 1998a). Chk1 mediates G2 arrest checkpoint pathway that prevents the onset of S phase by phosphorylating Cdc25 tyrosine phosphatase at a independently of the transcriptional function of p53 in serine residue, creating a binding site for 14-3-3, thus a vertebrate organism. inhibiting Cdc25 activity (Furnari et al., 1997; Sanchez et al., 1997; Zeng et al., 1998). Chk2, which is the vertebrate homolog of S. pombe Cds1 and S. cerevisiae Rad53, Introduction regulates p53 directly by phosphorylation (Chehab et al., 2000; Hirao et al., 2000; Shieh et al., 2000). In yeast, Cells respond to DNA damage by activating checkpoint where p53 is not present, the ATM homologs Mec1/Tel1 pathways that delay progression through the cell cycle for S. cerevisiae and Rad3 for S. pombe function through (Hensey and Gautier, 1995; Elledge, 1996). These signal-Chk1 to regulate cell cycle arrest in G2 and are also ing pathways operate throughout the cell cycle. Activaimportant for G1 and S phase delay (Weinert, 1992; tion of a DNA damage checkpoint can induce G1, S, or Siede et al., 1993; Bentley et al., 1996). In all organisms, G2 phase delay. These surveillance mechanisms are the mechanisms by which S phase entry is prevented essential to maintain the integrity of the genome. They in an ATM/Mec1/Rad3-dependent fashion are poorly ensure that damaged DNA templates are neither repliunderstood. cated nor segregated to the daughter cells until repaired. In vertebrates, the molecular dissection of the G1 ar-Failure to monitor and to signal following DNA damage rest mechanism has mainly focused on studies of p53is a hallmark of cancer cells (Hartwell and Kastan, 1994). dependent pathways. Responses mediated by p53 require transcription and de novo protein synthesis and therefore are more appropriate for a nonacute response , et al. (2000). Purifica-15 by phenylalanine and an EcoRI restriction site. The 3Ј primer contained an XhoI site: 5Ј-GCGCGAATTCATGGAGAACTTCCAA tion and characterization of ATM from human placenta. A manganese-dependent, wortmannin-sensitive serine/threonine protein ki-AAGGTGGAAAAGATCGGAGAGGGCGCGTTCGGAG; 3Ј-GCGCCT CGAGTCAGAGTCGAAGATGGGGTACTGGC. nase. J. Biol. Chem. 275, 7803-7810. The PCR product was sequenced and subcloned into pFastBac1 Chaturvedi, P., Eng, W.K., Zhu, Y., Mattern, M.R., Mishra, R., Hurle, (GIBCO) using the EcoRI and XhoI sites. Baculovirus expression M.R., Zhang, X., Annan, R.S., Lu, Q., Faucette, L.F., et al. (1999). system BacToBac (GIBCO) was then used to generate viral genomic Mammalian Chk2 is a downstream effector of the ATM-dependent DNA encoding for Cdk2AF. Sf9 cells were transfected with viral DNA damage checkpoint pathway. Oncogene 18, 4047-4054. genomic DNA carrying Cdk2AF, and viruses were harvested from Chehab, N.H., Malikzay, A., Appel, M., and Halazonetis, T.D. (2000). the medium and used for subsequent infections. Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by Expression and Purification of Proteins from Insect Cells stabilizing p53. Genes Dev. 14, 278-288. Active Cdk2AF/CyclinE and Cdk2/CyclinE complexes were purified from Sf9 cells according to Harper et al. (1995). Briefly, 150 ml of A., Scott, S.P., Hobson, K., and Lavin, M.F. (1999). Chk1 comple-Sf9 cells (1 ϫ 10 6 cells/ml) were infected with Cdk2AF virus or ments the G2/M checkpoint defect and radiosensitivity of ataxia-Cdk2WT virus along with GST-CyclinE virus. After 48 hr, cells were telangiectasia cells. Oncogene 18, 249-256. harvested and lysed in buffer containing 20mM Tris-HCL (pH 8.0), 2 mM EDTA, 100 mM NaCl, 5 mM NaF, 30 mM p-nitrophenylphos-Chong, J.P., Mahbubani, H.M., Khoo, C.Y., and Blow, J.J. (1995). phate, 1 mM PMSF, 0.5% Nonidet P-40 (Harper et al., 1995). The Purification of an MCM-containing complex as a component of the lysate was diluted 4-fold with 20 mM HEPES (pH 7.5), 15 mM MgCl 2 , DNA replication licensing systems. Nature 375, 418-421. 5 mM EGTA, 1 mM DTT, 1 mM ATP, and protease/phosphatase Chong, J.P., Thommes, P., Rowles, A., Mahbubani, H.M., and Blow, inhibitors were incubated with 1 ml of glutathione-Sepharose (Phar-J.J. (1997). Characterization of the Xenopus replication licensing macia) for 60 min. The resin was extensively washed, and GSTsystem. Methods Enzymol. 283, 549-564. CyclinE/Cdk2WT or GST-CyclinE/Cdk2AF was eluted with 2 ml of Costanzo, V., Avvedimento, E.V., Gottesman, M.E., Gautier, J., and 50mM Tris-HCL (pH 8.0) containing 20 mM glutathione, 120 mM Grieco, D. (1999). Protein kinase A is required for chromosomal DNA NaCl plus protease and phosphatase inhibitors. replication. Curr. Biol. 9, 903-906. Xenopus Cdc25A, Cdc25A catalytic inactive mutant (C432A), and Dasso, M., and Newport, J.W. (1990). Completion of DNA replication Cdc25C were purified according to Izumi and Maller (1993). Cdc25is monitored by a feedback system that controls the initiation of expressing baculoviruses were a gift from Dr. J. Maller. mitosis in vitro: studies in Xenopus. Cell 61, 811-823. Elledge, S.J. (1996). Cell cycle checkpoints: preventing an identity
Checkpoint Control of DNA Repair in Yeast
2021
Budding yeast has been a model organism for understanding how DNA damage is repaired and how cells minimize genetic instability caused by arresting or delaying the cell cycle at well-defined checkpoints. However, many DNA damage insults are tolerated by mechanisms that can both be error-prone and error-free. The mechanisms that tolerate DNA damage and promote cell division are less well-understood. This review summarizes current information known about the checkpoint response to agents that elicit both the G2/M checkpoint and the intra-S phase checkpoint and how cells adapt to unrepaired DNA damage. Tolerance to particular bulky DNA adducts and radiomimetic agents are discussed, as well as possible mechanisms that may control phosphatases that deactivate phosphorylated proteins.
Genes & Development, 1994
In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The Gz-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MECl and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13-, cdcl3 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdcl3 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arrest of cells before mitosis.
Induction of a G1-S checkpoint in fission yeast
Proceedings of the National Academy of Sciences, 2012
Entry into S phase is carefully regulated and, in most organisms, under the control of a G 1-S checkpoint. We have previously described a G 1-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G 1-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G 1-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G 1-S delay depends on at least two different inputs.
DNA damage checkpoint maintenance through sustained Chk1 activity
Journal of Cell Science, 2004
The G2 DNA damage checkpoint prevents mitotic entry in the presence of DNA damage. This requires the activation of the phosphoinositide-3-kinase-related protein kinases ATR and ATM in human cells and the ATR homologue Rad3 in the fission yeast Schizosaccharomyces pombe. Rad3 activates the effector protein kinase Chk1 by phosphorylation. However, in fission yeast, inactivation of Rad3 following checkpoint activation has no impact on checkpoint duration. This demonstrates that Rad3 is not required for checkpoint maintenance and that the processes of checkpoint initiation and maintenance are distinct. Chk1 is required for checkpoint initiation but its role in checkpoint maintenance has not been investigated. We show here that Chk1 kinase activity is rapidly induced following irradiation and is maintained for the duration of a checkpoint arrest. On entry to mitosis, there is a transient decrease in Chk1 activity and phosphorylation, but Chk1 activity remains higher than that observed in...