scispace - formally typeset
Search or ask a question
Journal ArticleDOI

DNA-replication checkpoint control at the Drosophila midblastula transition

03 Jul 1997-Nature (Nature Publishing Group)-Vol. 388, Iss: 6637, pp 93-97
TL;DR: This article showed that mutations in the grapes (grp) checkpoint 1 kinase homologue in Drosophila block the morphological and biochemical changes that accompany the midblastula transition, leading to a continuation of the maternal cell-cycle programme, and disrupt DNA-replication checkpoint control of cell cycle progression.
Abstract: Embryogenesis is typically initiated by a series of rapid mitotic divisions that are under maternal genetic control. The switch to zygotic control of embryogenesis at the midblastula transition is accompanied by significant increases in cell-cycle length and gene transcription, and changes in embryo morphology. Here we show that mutations in the grapes (grp) checkpoint 1 kinase homologue in Drosophila block the morphological and biochemical changes that accompany the midblastula transition, lead to a continuation of the maternal cell-cycle programme, and disrupt DNA-replication checkpoint control of cell-cycle progression. The timing of the midblastula transition is controlled by the ratio of nuclei to cytoplasm (the nucleocytoplasmic ratio), suggesting that this developmental transition is triggered by titration of a maternal factor by the increasing mass of nuclear material that accumulates during the rapid embryonic mitoses. Our observations support a model for cell-cycle control at the midblastula transition in which titration of a maternal component of the DNA-replication machinery slows DNA synthesis and induces a checkpoint-dependent delay in cell-cycle progression. This delay may allow both completion of S phase and transcription of genes that initiate the switch to zygotic control of embryogenesis.

Content maybe subject to copyright    Report

Citations
More filters
Journal ArticleDOI
TL;DR: It is shown that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU).
Abstract: Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G(2)/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.

1,707 citations

Journal ArticleDOI
05 Sep 1997-Science
TL;DR: Results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry.
Abstract: In response to DNA damage, mammalian cells prevent cell cycle progression through the control of critical cell cycle regulators. A human gene was identified that encodes the protein Chk1, a homolog of the Schizosaccharomyces pombe Chk1 protein kinase, which is required for the DNA damage checkpoint. Human Chk1 protein was modified in response to DNA damage. In vitro Chk1 bound to and phosphorylated the dual-specificity protein phosphatases Cdc25A, Cdc25B, and Cdc25C, which control cell cycle transitions by dephosphorylating cyclin-dependent kinases. Chk1 phosphorylates Cdc25C on serine-216. As shown in an accompanying paper by Peng et al. in this issue, serine-216 phosphorylation creates a binding site for 14-3-3 protein and inhibits function of the phosphatase. These results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry.

1,383 citations

Journal ArticleDOI
TL;DR: It is demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1, an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation.
Abstract: Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.

1,065 citations

Journal ArticleDOI
TL;DR: Targeted disruption of Chk1 in mice showed that ChK1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage, which may indicate that Chk 1 is indispensable for cell proliferation and survival through maintaining the G(2) checkpoint in mammals.
Abstract: The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed that Chk1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1(-/-) blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis in Chk1(-/-) embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G(2) checkpoint in mammals.

521 citations


Cites background from "DNA-replication checkpoint control ..."

  • ...In addition, a Drosophila Chk1 homolog, Grapes, modulates cell cycle timing at the midblastula transition during embryogenesis (Sibon et al. 1997)....

    [...]

  • ...This idea is supported by the fact that the Drosophila Chk1 mutant, Grapes (Sibon et al. 1997), and the ATR homolog, Mei-41, mutant (Sibon et al. 1999) die at the midblastula transition, showing a defect in DNA replication checkpoint function....

    [...]

Journal ArticleDOI
TL;DR: It is suggested that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.
Abstract: The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.

440 citations


Cites background from "DNA-replication checkpoint control ..."

  • ...Likewise, the Drosophila Chk1 homolog Grapes is required for the replication checkpoint in early embryos (Fogarty et al. 1997; Sibon et al. 1997)....

    [...]

References
More filters
Journal ArticleDOI
TL;DR: A non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos and revealed translational control of the maternally derived hb mRNA, which was difficult to detect by conventional techniques.
Abstract: We have developed a non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos. After fixation, whole embryos are hybridized in situ with a DNA probe which has been labeled with digoxygenin. The hybridization products are detected by using a phosphatase-coupled antibody against digoxygenin. In parallel experiments, embryos can be treated with an antibody directed against the corresponding protein product to allow the detection of its distribution using standard immunochemical techniques. We have used this approach to compare the spatial and temporal distribution patterns of the RNA and protein products of the segmentation gene hunchback (hb) during the early stages of embryogenesis. This comparison revealed translational control of the maternally derived hb mRNA, which was difficult to detect by conventional techniques. The non-radioactive in situ hybridization method is as sensitive as conventional methods, but is faster and easier to perform. This may make it a useful tool for a variety of other systems.

2,327 citations

Journal ArticleDOI
06 Dec 1996-Science
TL;DR: Signal transduction pathways that transmit checkpoint signals in response to DNA damage, replication blocks, and spindle damage are revealed, underscoring the conservation of cell cycle regulatory machinery.
Abstract: Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity. In addition, checkpoints respond to damage by arresting the cell cycle to provide time for repair and by inducing transcription of genes that facilitate repair. Checkpoint loss results in genomic instability and has been implicated in the evolution of normal cells into cancer cells. Recent advances have revealed signal transduction pathways that transmit checkpoint signals in response to DNA damage, replication blocks, and spindle damage. Checkpoint pathways have components shared among all eukaryotes, underscoring the conservation of cell cycle regulatory machinery.

2,114 citations


"DNA-replication checkpoint control ..." refers background in this paper

  • ...Studies in yeast and vertebrates indicate that the Cdc2–cyclin complex is activated by the Cdc25 phosphatase, which removes the inhibitory tyrosine phosphates and thus drives the cell into mitosi...

    [...]

Journal ArticleDOI
01 Oct 1982-Cell
TL;DR: The Xenopus embryo undergoes 12 rapid synchronous cleavages followed by a period of slower asynchronous divisions more typical of somatic cells, termed the midblastula transition (MBT), which shows that at the MBT the blastomeres become motile and transcriptionally active for the first time.

1,587 citations


"DNA-replication checkpoint control ..." refers background in this paper

  • ...The switch to zygotic control of embryogenesis at the midblastula transition is accompanied by significant increases in cell-cycle length and gene transcription, and changes in embryo morpholog...

    [...]

Journal ArticleDOI
TL;DR: Using differential interference contrast optics, combined with cinematography, the morphological changes that the living, syncytial embryo undergoes from stage 10 through 14 of Drosophila embryogenesis, that is just prior to and during formation of the cellular blastoderm are studied.
Abstract: Using differential interference contrast optics, combined with cinematography, we have studied the morphological changes that the living, syncytial embryo undergoes from stage 10 through 14 of Drosophila embryogenesis, that is just prior to and during formation of the cellular blastoderm. We have supplemented these studies with data collected from fixed, stained, whole embryos. The following information has been obtained. The average duration of nuclear cycles 10, 11, 12 and 13 is about 9, 10, 12 and 21 min, respectively (25 degrees C). In these four cycles, the duration of that portion of the mitotic period that lacks a discrete nuclear envelope is 3, 3, 3 and 5 min, respectively. The length of nuclear cycle 14 varies in a position-specific manner throughout the embryo, the shortest cycles being of 65 min duration. During nuclear cycles 10 through 13, it is commonly observed in living embryos that the syncytial blastoderm nuclei enter (and leave) mitosis in one of two waves that originate nearly simultaneously from the opposite anterior and posterior poles of the embryo, and terminate in its midregion. From our preparations of quick-frozen embryos, we estimate that these mitotic waves take on average about half a minute to travel over the embryonic surface from pole to equator. The yolk nuclei, which remain in the core of the embryo when the rest of the nuclei migrate to the periphery, divide in synchrony with the migrating nuclei at nuclear cycles 8 and 9, and just after the now peripherally located nuclei at nuclear cycle 10. After cycle 10, these yolk nuclei cease dividing and become polyploid. The syncytial embryo has at least three distinct levels of cytoskeletal organization: structured domains of cytoplasm are organized around each blastoderm nucleus; radially directed tracks orient colchicine-sensitive saltatory transport throughout the peripheral cytoplasm; and a long-range organization of the core of the embryo makes possible coherent movements of the large inner yolk mass in concert with each nuclear cycle. This highly organized cytoplasm may be involved in providing positional information for the important process of nuclear determination that is known to occur during these stages.

1,035 citations