Proteins in the Nutrient-Sensing and DNA Damage Checkpoint Pathways Cooperate to Restrain Mitotic Progression following DNA Damage (original) (raw)
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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...
Molecular Cell, 2001
In budding yeast, a single unrepaired DSB, for example, one created by expression of the site-specific HO endonuclease, is sufficient to cause G2/M arrest (Sandell and Zakian, 1993). However, these cells do not remain permanently arrested; rather, after 8-12 hr they resume progression through the cell cycle (Sandell and Waltham, Massachusetts 02454 Zakian, 1993; Toczyski et al., 1997; Lee et al., 1998). This † Istituto F.I.R.C. di Oncologia Molecolare and escape from G2/M arrest occurs despite the continued Dipartimento di Genetica e di Biologia dei presence of the broken chromosome and hence is Microrganismi termed adaptation (Sandell and Zakian, 1993; Toczyski Universita' degli Studi di Milano et al., 1997; Lee et al., 1998).
Molecular and Cellular Biology, 2004
DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors. Here we show that the Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage. Both cell cycle-and DNA damage-dependent Sae2 phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3. Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1. Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions.
Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint
Molecular Biology of …, 2000
The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in ⌬mik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint. . (1994). Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. Mol. Biol. Cell 5, 147-160. Figure 9. Model of the DNA damage checkpoint in fission yeast.
The DNA Damage Checkpoint Signal in Budding Yeast Is Nuclear Limited
Molecular Cell, 2000
and of several key components is regulated; for example, in Department of Genetics mammalian cells Cdk1 is prevented from entering the Stanford University nucleus in response to DNA damage by a 14-3-3 protein Stanford, California 94305 (Jin et al., 1998; Chan et al., 1999). In fission yeast, † Rosenstiel Center and although Cdk1 seems to be predominantly nuclear at Department of Biology all times, the localization of its activating phosphatase, Brandeis University Cdc25, changes in response to DNA damage, becoming Waltham, Massachusetts 02454 cytoplasmic (Furnari et al., 1999). These observations suggest that control of the entry into mitosis after DNA damage might require transmission of the DNA damage Summary signal to the cytoplasmic compartment, although they could also be explained by a nuclear-limited active ex-The nature of the DNA damage-induced checkpoint port of a key mitotic component from the nucleus. signal that causes the arrest of cells prior to mitosis In the budding yeast Saccharomyces cerevisiae, inis unknown. To determine if this signal is transmitted hibitory phosphorylation of Cdc28p, the homolog of Cdk1, does not play a role in cell cycle arrest after DNA through the cytoplasm or is confined to the nucleus, damage (Amon et al., 1992; Sorger and Murray, 1992).
Genes & Development, 1996
SPK1/RAD53/MEC2/SAD1 of Saccharomyces cerevisiae encodes an essential protein kinase that is required for activation of replication-sensitive and DNA damage-sensitive checkpoint arrest. We have investigated the regulation of phosphorylation and kinase activity of Spklp during the cell cycle and by conditions that activate checkpoint pathways. Phosphorylation of Spklp is induced by treatment of cells with agents that damage DNA or interfere with DNA synthesis. Although only S-and Ge-phase cdc mutants arrest with hyperphosphorylated Spklp, damage-induced phosphorylation of Spklp can occur in G 1 and M as well.
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...
Proceedings of the National Academy of Sciences, 2005
Mitotic catastrophe is the response of mammalian cells to mitotic DNA damage. It produces tetraploid cells with a range of different nuclear morphologies from binucleated to multimicronucleated. In response to DNA damage, checkpoints are activated to delay cell cycle progression and to coordinate repair. Cells in different cell cycle phases use different mechanisms to arrest their cell cycle progression. It has remained unclear whether the termination of mitosis in a mitotic catastrophe is regulated by DNA damage checkpoints. Here, we report the presence of a mitotic exit DNA damage checkpoint in mammalian cells. This checkpoint delays mitotic exit and prevents cytokinesis and, thereby, is responsible for mitotic catastrophe. The DNA damage-induced mitotic exit delay correlates with the inhibition of Cdh1 activation and the attenuated degradation of cyclin B1. We demonstrate that the checkpoint is Chk1-dependent.
Mechanisms of Ageing and Development, 2009
Stationary phase Saccharomyces cerevisiae can serve as a model for postmitotic cells of higher eukaryotes. Phosphorylation and activation of the checkpoint kinase Rad53 was observed after more than 2 days of culture if two major pathways of oxidative DNA damage repair, base excision repair (BER) and nucleotide excision repair (NER), are inactive. The wild type showed a low degree of Rad53 phosphorylation when the incubation period was drastically increased. In the ber ner strain, Rad53 phosphorylation can be abolished by inclusion of antioxidants or exclusion of oxygen. Furthermore, this modification and enhanced mutagenesis in extended stationary phase were absent in rho° strains, lacking detectable mitochondrial DNA. This checkpoint response is therefore thought to be dependent on reactive oxygen species originating from mitochondrial respiration. There was no evidence for progressive overall telomere shortening during stationary-phase incubation. Since Rad50 (of the MRN complex) and Mec1 (the homolog of ATR) were absolutely required for the observed checkpoint response, we assume that resected random double-strand breaks are the critical lesion. Single-strand resection may be accelerated by unrepaired oxidative base damage in the vicinity of a double-strand break.