grapes: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - grapes

Synonyms -

Cytological map position - 36A10--36A14

Function - cell cycle checkpoint protein

Keywords - cell cycle, response to DNA damage

Symbol - grp

FlyBase ID:FBgn0261278

Genetic map position -

Classification - Chk1 homolog - kinase

Cellular location - presumably nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Deneke, V.E., Melbinger, A., Vergassola, M. and Di Talia, S. (2016). Waves of Cdk1 activity in S phase synchronize the cell cycle in Drosophila embryos. Dev Cell 38: 399-412. PubMed ID: 27554859
Summary:
Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. Using biosensors of Cdk1 and Chk1 activities, this study dissected the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. Cdk1 waves were shown not to be controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. S phase Cdk1 waves were shown to be fundamentally distinct and propagate as active trigger waves in an excitable medium, while mitotic Cdk1 waves propagate as passive phase waves. These findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. 

Lefebvre, F. A., Benoit Bouvrette, L. P., Bergalet, J. and Lecuyer, E. (2017). Biochemical fractionation of time-resolved Drosophila embryos reveals similar transcriptomic alterations in replication checkpoint and histone mRNA processing mutants. J Mol Biol [Epub ahead of print]. PubMed ID: 28167048
Summary:
In higher eukaryotes, maternally provided gene products drive the initial stages of embryogenesis until the zygotic transcriptional program takes over, a developmental process called the midblastula transition (MBT). In addition to zygotic genome activation, the MBT involves alterations in cell-cycle length and the implementation of DNA damage/replication checkpoints that serve to monitor genome integrity. Previous work has shown that mutations affecting histone mRNA metabolism or DNA replication checkpoint factors severely impact developmental progression through the MBT, prompting this study to characterize and contrast the transcriptomic impact of these genetic perturbations. This study defined gene expression profiles that mark early embryogenesis in Drosophila through transcriptomic analyses of developmentally staged (early syncytial vs late blastoderm) and biochemically fractionated (nuclear vs cytoplasmic) wild type embryos. The transcriptomic profiles were compared of loss-of-function mutants of the Chk1/Grapes replication checkpoint kinase and the Stem Loop Binding Protein (SLBP), a key regulator of replication-dependent histone mRNAs. This analysis of RNA spatial and temporal distribution during embryogenesis offers new insights into the dynamics of early embryogenesis. In addition, it was found that grp and Slbp mutant embryos display profound and highly similar defects in gene expression, most strikingly in zygotic gene expression, compromising the transition from a maternal to a zygotic regulation of development.
Kizhedathu, A., Bagul, A. V. and Guha, A. (2018). Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila. Elife 7. PubMed ID: 29658881
Summary:
Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. This study investigated the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). Tracheoblasts were shown to pause in G2 for ~48-56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). Mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24-32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. It is proposed that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.
BIOLOGICAL OVERVIEW

Treatment of cells with radiation causes the cell cycle to pause in the G2 phase, thus stopping cells from proceeding through mitosis. In addition, when the G2 phase is shortened for any reason, cells become sensitive to radiation. These observations have led to the concept of "checkpoints" - specific times within the cell cycle during which progression through the cycle can be delayed in response to either DNA damage or to the incompletion of prior cell cycle events, such as DNA replication. Grapes protein, a Drosophila homolog of the Chk1 protein of fission yeast, is involved in a developmentally regulated interphase checkpoint during the late syncytial divisions in early embryogenesis. This delay may function to allow both the completion of S phase and the transcription of genes that initiate the switch to zygotic control of embryogenesis (Sibon, 1997 and Fogarty, 1997). grapes mutation causes a metaphase arrest at nuclear cycle 13, early in embryogenesis; grapes mutants undergo cessation of cell cycles upon irradiation of syncytial embryos (Fogarty, 1994). This stage in Drosophila development is termed the midblastula transition, and takes place during blastoderm cellularization. It is marked by the initiation of zygotic transcription. Before describing Grapes in detail, the biochemical pathways for the regulation of G2-M checkpoint control will be described in some detail, since Grapes function in Drosophila is part of an conserved pathway for mitotic control that is present in many species, from yeast to mammals.

Many proteins active in checkpoint pathways have been identified by genetic analysis of yeast, and their mammalian counterparts have also been identified. Mitotic checkpoints require three distinct functions: a detection system to determine the change in DNA structure; a signal pathway to transmit this information; and an effector mechanism to interact with the cell cycle machinery (Carr, 1996). One effector, Chk1, is required in yeast for mitotic arrest after DNA damage. Chk1 is phosphorylated in response to DNA damage and has been shown in various studies to target Wee1 kinase or Cdc2 in the DNA damage checkpoint, Wee 1 kinase (see Drosophila wee) inhibits the cyclin cdc2 (see Drosophila cdc2) by phosphorylation on tyrosine 15, preventing the G2-M transition (O'Connell, 1997).

How is Chk1 phosphorylation regulated? What are the upstream components of DNA damage control? Experimental evidence points to the involvement of two families of proteins. The first family consists of BRCT domain proteins. The conserved BRCT domain, a protein interaction motif, was first described at the carboxyl terminus of the breast cancer protein BRCA1, a p53 binding protein (BP531), and the yeast cell cycle checkpoint protein RAD9. Because BRCT proteins are often quite large, they are almost certainly multifunctional proteins. Many of them could function as scaffolding proteins that assemble into multiple protein complexes at the site of DNA damage and many may not serve as the immediate sensors of DNA damage. Functional domains found in different BRCT domain proteins include DNA binding domains, DNA ligases, PCNA binding domains, RNA binding domains, ankyin repeats and ring fingers (Bork, 1997).

A second family associated with upstream components of checkpoint pathways includes the ATM protein deficient in ataxia-telangiectasia patients rendering them sensitive to ionizing radiation. Sharing structural homology with Atm is the Atr (for ataxia-telangiectaia- and rad3-related) protein and related yeast proteins Rad3/Mec1 and Tel1. In Drosophila, mei-41 encodes an Atr homolog and mei-41 flies have reduced meiotic recombination frequencies and aberrant recombination nodules (Hari, 1995). Atm protein is similar to a family of phosphatidylinositol 3-kinases. Members of this family, which includes the DNA-dependent protein kinase catalytic subunit, appear to act as sensors for defects in various cell-cycle transitions or aberrations in chromosomal mechanisms. Mec1 from S. cerevisiae, Rad3 from S. pombe, and Mei-41 from Drosophila appear to be structural and functional homologs that play important roles in mitotic and meiotic cell-cycle checkpoints and DNA repair. These three proteins play roles in G2 checkpoint control in mitotic cells and also play important roles in meiosis, suggesting important functional links between the two processes (Keegan, 1996 and references).

Proteins of both Rad3 and BRCT related families associate with meiotic chromosomes. Meiosis segregates a complete haploid set of chromosomes to each gamete, and this process requires the negotiation of a complex series of interdependent events. Homologous chromosomes must associate, recombine, synapse, and segregate to opposite poles at meiosis I. Although events are less clear in multicellular organisms, DNA double-stranded breaks have been linked to the initiation of meiotic chromosomal synapse in yeast. Atr is localized to the nuclei of primary spermatocytes, cells that are undergoing meiosis I. Both Atr and Atm proteins are present at pairing forks in the meiotic chromosomes of mice, however, they do not colocalize; instead they occupy complementary positions. Atr localizes along unsynapsed chromosome axes and Atm interacts with synapsed axes (Keegan, 1996). The presence of Chk1 along meiotic chromosomes is dependent on Atm (Flaggs, 1997). BRCA1, found in discrete, nuclear foci during S phase of the mitotic cycle is detected on unsynapsed elements of human synaptonemal complexes. BRCA1 associates with Rad51, a homolog of bacterial RecA. Rad51 is a member of a protein family known to mediate DNA strand-exchange functions leading to normal recombination. The association of Rad3 and BRCT family proteins with meiotic chromosomes and the fact that mutations in genes coding for these proteins are involved in defects in DNA damage-induced cell cycle responses, radiation hypersensitivity, and defective meiosis, suggests a role for these proteins in one or more signal transduction cascades involved in meiotic and mitotic checkpoint control (Scully, 1997).

grapes, coding for the Drosophila Chk1 homolog, was identified as a member of a set of maternal-effect mutations in which the affected embryos undergo abnormal divisions only after the nuclei have migrated to the cortex. Each of these mutations uniquely and specifically disrupts the cortical cytoskeleton. In grp mutants, during anaphase and telophase of nuclear cycle 12, the spindle deteriorates and the usually pronounced midbody (the microtubular structure separating chromosomes) never forms. Without the midbody, sister telophase nuclei snap-back and fuse. The fused telophase nuclei undergo an abbreviated nuclear cycle 13 interphase, fail to properly condense their chromosomes during prophase, and arrest in metaphase. X-irradiation experiments demonstrate that the grp induced arrest is not a direct consequence of processes specific to nuclear cycle 13. In normal embryos, X-irradiation induces a high error rate but no arrest. In contrast, X-irradiation of grp-derived embryos results in a metaphase arrest even during the pre-migration and initial cortical nuclear cycles, prior to nuclear cycle 13 (Fogarty, 1994).

During the final syncytial divisions before the midblastula transition (divisions 11-13), the duration of interphase increases from 6 to 14 minutes. Interphase 14 is prolonged, lasting over 60 minutes, during which time invaginating membranes surround the majority of nuclei to form a cellular blastoderm. Embryos deprived of grapes do not show a significant increase in interphase length during syncytial divisions 11-13. After mitosis 13, mutant embryos proceed through at least two extra syncytial cycles of spindle assembly and disassembly, and nuclear envelope breakdown and formation; cellularization is never observed. Thus grapes mutation blocks nearly all the changes in cell-cycle dynamics and embryo morphology associated with the midblastula transition (Sibon, 1997).

The changes in cell-cycle length are associated with changes in phosphorylation of the cyclin-dependent kinase cdc2. Termination of the rapid syncytial divisions is accompanied by accumulation of a low electorphoretic-mobility inhibitory tyrosine-phosphorylated form of Cdc2. In string mutants, this tyrosine-phosphorylated form of Cdc2 does not accumulate. It is known that the Cdc2-cyclin complex is activated by the Cdc25 phosphatase known in Drosophila as String, which removes the inhibitory tyrosine phosphates and thus drives the cell into mitosis. In Drosophila, a dramatic decrease in the level of String accompanies the increase in cell-cycle time at the midblastula transition. In grapes mutants, String protein levels do not decrease significantly during the later syncytial mitoses. As Chk1 is known to target wee1 kinase in yeast (O'Connell, 1997), which in turn targets cdc2 in yeast and Drosophila (Campbell, 1995), it is likely that Chk1 targets Wee kinase in Drosophila which in turn maintains cdc2 in an inactive state.

A simple DNA-replication checkpoint model is suggested for cell-cycle control at the midblastula transition. In his model, a free-running cell-cycle oscillator drives the rapid divisions that initiate embryogenesis. During the first 10 divisions, maternally supplied components of the DNA synthesis/initiation machinery are in excess, and S phase is completed before the oscillator triggers mitosis. During divisions 10-13, in contrast, a component of the maternal replication machinery is titrated by the increasing mass of nuclear DNA and becomes rate limiting. DNA synthesis therefore slows, and S phase cannot be completed before the free-running cell-cycle oscillator would normally trigger mitosis. A Grapes kinase-dependent checkpoint pathway is therefore activated and delays M phase until DNA synthesis is complete. This model is consistent with regulation of the midblastula transition by the nucleocytoplasmic ratio, and explains the absence of a G2 phase and the increase in S phase during the later syncytial mitoses, and accounts for the cell-cycle timing defects observed in grp mutant embryos (Sibon, 1997). This model does not take into account the likelihood that GRP mRNA is actively degraded at the midblastula transition, suggesting that additional regulation of transcript level is involved in the disappearance of Grapes during the transition (Fogarty, 1997).

grp (chk1) replication-checkpoint mutations and DNA damage trigger a Chk2-dependent block at the Drosophila midblastula transition

The 13 syncytial cleavage divisions that initiate Drosophila embryogenesis are under maternal genetic control. The switch to zygotic regulation of development at the midblastula transition (MBT) follows mitosis 13, when the cleavage divisions terminate, transcription increases and the blastoderm cellularizes. Embryos mutant for grp, which encodes Checkpoint kinase 1 (Chk1), are DNA-replication-checkpoint defective and fail to cellularize, gastrulate or to initiate high-level zygotic transcription at the MBT. The mnk (also known as loki) gene encodes Checkpoint kinase 2 (Chk2), which functions in DNA-damage signal transduction. mnk grp double-mutant embryos are replication-checkpoint defective but cellularize, gastrulate and activate high levels of zygotic gene expression. grp mutant embryos accumulate DNA double-strand breaks and DNA-damaging agents induce a mnk-dependent block to cellularization and zygotic gene expression. It is concluded that the DNA-replication checkpoint maintains genome integrity during the cleavage divisions, and that checkpoint mutations lead to DNA damage that induces a novel Chk2-dependent block at the MBT (Takada, 2007).

Studies in lower eukaryotes initially defined checkpoints as< non-essential pathways that delay cell cycle progression in response to external stress. Subsequently, checkpoint mutations in higher eukaryotes were found to induce developmental defects and embryonic lethality. In Drosophila, the DNA-replication checkpoint is required to delay the cell cycle during the late cleavage stage, and checkpoint mutants subsequently fail to cellularize or activate zygotic gene expression at the MBT. These findings suggested that the replication checkpoint has a direct role in metazoan developmental. Alternatively, the observed developmental defects could be an indirect consequence of checkpoint failure (Takada, 2007).

A null mutation in mnk, which encodes the conserved DNA-damage signaling kinase Chk2, efficiently suppresses the cellularization and zygotic gene-activation defects in grp, but does not restore wild-type cell cycle timing or replication-checkpoint function. It is therefore concluded that progression through the Drosophila MBT does not directly require Chk1 or checkpoint-dependent cell cycle delays. Instead, the data indicate that the essential function for the replication checkpoint is to prevent DNA damage during the syncytial blastoderm divisions, which triggers a Chk2-dependent block to zygotic gene activation and cellularization. Supporting this proposal, DNA-damaging agents trigger a Chk2-dependent block to cellularization and zygotic gene activation, and grp mutations accumulate DNA double-strand breaks. Chk2 is likely to have multiple targets during this developmental response to DNA damage; these targets may include transcription factors that control the expression of genes implicated in cell cycle control and cellularization (Takada, 2007).

Embryos mutant for grp or mei-41 lack a functional replication checkpoint and progress into mitosis prior to S-phase completion, triggering defects in γ-Tubulin localization and microtubule nucleation. These mitotic defects are suppressed by mnk, raising the possibility that mnk suppresses the grp mutant developmental block at the MBT by restoring mitotic function. However, mnk does not suppress the chromosome-segregation defects associated with grp mutants. More significantly, inducing DNA damage following the final syncytial blastoderm division triggers a Chk2-dependent block to cellularization. DNA damage can therefore induce a Chk2-dependent developmental block that is distinct from the damage and Chk2-dependent block to mitosis (Takada, 2007).

The studies outlined here support a simple model in which the developmental arrest associated with grp mutations results from defects in the established function for this kinase in cell cycle control. The early cleavage-stage divisions have a simplified S-phase/M-phase cell cycle, and it is proposed that the crucial function of Chk1 is to delay mitosis until DNA replication is complete. In grp mutants, progression into mitosis before replication is complete leads to DNA damage, which activates a Chk2-dependent block to developmental progression. Intriguingly, disrupting Chk1 function also leads to early embryonic lethality in frogs, mice and worms. Chk1 knockdown in Xenopus and Chk1 (also known as Chek1 - Mouse Genome Informatics) mutations in mouse lead to apoptotic death of the embryo, consistent with a DNA-damage response. It is therefore speculated that Chk1 has a conserved function in maintaining genome integrity during the cleavage stage, and that the early embryonic lethality in checkpoint mutants is a consequence of DNA-damage signaling (Takada, 2007).


GENE STRUCTURE

Sequencing of additional grp cDNAs reveals differences in the 5' untranslated regions. These differences are presumably due to alternative splicing or alternative promoter usage at the grp locus, but do not affect the translation product (Fogarty, 1997).

cDNA clone length - 2567 bases

Bases in 5' UTR - 737

Bases in 3' UTR - 290


PROTEIN STRUCTURE

Amino Acids - 513

Structural Domains

Grapes has extensive structural similarity to the catalytic domain of the serine/threonine family of protein kinases. One member of this family, the Chk1/Rad27 DNA damage checkpoint kinase of S. pombe has significant homology to the predicted Grp protein, extending beyond the kinase domain. Over a contiguous 97 amino acid region within the kinase domain, the proteins exhibit 59% identity and 82% conservation. Over the entire protein sequence, the two proteins exhibit 26% amino acid identity. Outside the kinase domain, five regions, ranging in length from 9 to 24 amino acids, exhibit a greater than 30% amino acid sequence identity (Fogarty, 1997).


grapes: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 February 98

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