The Spd1p S phase inhibitor can activate the DNA replication checkpoint pathway in fission yeast

2000 ◽  
Vol 113 (23) ◽  
pp. 4341-4350 ◽  
Author(s):  
A. Borgne ◽  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
A Cell ◽  
Checkpoint Genes

Spd1p (for S phase delayed) is a cell cycle inhibitor in Schizosaccharomyces pombe. Spd1p overexpression blocks the onset of both S phase and mitosis. In this study, we have investigated the mechanisms by which Spd1p overexpression blocks cell cycle progression, focussing on the block over mitotic onset. High levels of Spd1p lead to an increase in Y15 phosphorylation of Cdc2p and we show that the block over G(2) requires the Wee1p kinase and is dependent on the rad and chk1/cds1 checkpoint genes. We propose that high levels of Spd1p in G(2) cells activate the DNA replication checkpoint control, which leads to a Wee1p-dependent increase of Cdc2p Y15 phosphorylation blocking onset of mitosis. The Spd1p block at S phase onset may act by interfering directly with DNA replication, and also activates the G(2)rad/hus checkpoint pathway to block mitosis.

Cdc18p can block mitosis by two independent mechanisms

1998 ◽  
Vol 111 (20) ◽  
pp. 3101-3108 ◽  
Author(s):  
E. Greenwood ◽  
H. Nishitani ◽  
P. Nurse
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Mitotic Kinase ◽  
C Terminus ◽  
N Terminus ◽  
Dna Replication Checkpoint ◽  
Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

A method for assaying the sensitivity of Drosophila replication checkpoint mutants to anti-cancer and DNA-damaging drugs.

Genome ◽  
2002 ◽  
Vol 45 (5) ◽  
pp. 881-889 ◽  
Author(s):  
Colleen M Radcliffe ◽  
Elizabeth A Silva ◽  
Shelagh D Campbell
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Tumor Formation ◽  
Cycle Progression ◽  
Replication Checkpoint ◽  
Anti Cancer ◽  
Dna Replication Checkpoint ◽  
Checkpoint Genes ◽  
Regulate Cell Cycle Progression

In multi-cellular organisms, failure to properly regulate cell-cycle progression can result in inappropriate cell death or uncontrolled cell division leading to tumor formation. To guard against such events, conserved regulatory mechanisms called "checkpoints" block progression into mitosis in response to DNA damage and incomplete replication, as well as in response to other signals. Checkpoint mutants in organisms as diverse as yeast and humans are sensitive to various chemical agents that inhibit DNA replication or cause DNA damage. This phenomenon is the primary rationale for chemotherapy, which uses drugs that preferentially target tumor cells with compromised checkpoints. In this study, we demonstrate the use of Drosophila checkpoint mutants as a system for assaying the effects of various DNA-damaging and anti-cancer agents in a developing multicellular organism. Dwee1, grp and mei-41 are genes that encode kinases that function in the DNA replication checkpoint. We tested zygotic mutants of each gene for sensitivity to the DNA replication inhibitor hydroxyurea (HU), methyl methanosulfonate (MMS), ara-C, cisplatin, and the oxygen radical generating compound paraquat. The mutants show distinct differences in their sensitivity to each of the drugs tested, suggesting an underlying complexity in the responses of individual checkpoint genes to genotoxic stress.Key words: hydroxyurea (HU), ara-C, cisplatin, methyl methane sulfonate (MMS), paraquat.

scholarly journals The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci

2000 ◽  
Vol 14 (1) ◽  
pp. 81-96 ◽  
Author(s):  
Christian Frei ◽  
Susan M. Gasser
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
S Phase ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Replication Fork Progression ◽  
Cycle Arrest ◽  
Dna Replication Checkpoint

We have examined the cellular function of Sgs1p, a nonessential yeast DNA helicase, homologs of which are implicated in two highly debilitating hereditary human diseases (Werner's and Bloom's syndromes). We show that Sgs1p is an integral component of the S-phase checkpoint response in yeast, which arrests cells due to DNA damage or blocked fork progression during DNA replication. DNA polε and Sgs1p are found in the same epistasis group and act upstream of Rad53p to signal cell cycle arrest when DNA replication is perturbed. Sgs1p is tightly regulated through the cell cycle, accumulates in S phase and colocalizes with Rad53p in S-phase-specific foci, even in the absence of fork arrest. The association of Rad53p with a chromatin subfraction is Sgs1p dependent, suggesting an important role for the helicase in the signal-transducing pathway that monitors replication fork progression.

scholarly journals DNA replication times the cell cycle and contributes to the mid-blastula transition in Drosophila embryos

2009 ◽  
Vol 187 (1) ◽  
pp. 7-14 ◽  
Author(s):  
Mark L. McCleland ◽  
Antony W. Shermoen ◽  
Patrick H. O'Farrell
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cycle Progression ◽  
Replication Checkpoint ◽  
Drosophila Embryogenesis ◽  
Zygotic Transcription ◽  
Phase Cycle ◽  
Drosophila Embryos

We examined the contribution of S phase in timing cell cycle progression during Drosophila embryogenesis using an approach that deletes S phase rather than arresting its progress. Injection of Drosophila Geminin, an inhibitor of replication licensing, prevented subsequent replication so that the following mitosis occurred with uninemic chromosomes, which failed to align. The effect of S phase deletion on interphase length changed with development. During the maternally regulated syncytial blastoderm cycles, deleting S phase shortened interphase, and deletion of the last of blastoderm S phase (cycle 14) induced an extra synchronous division and temporarily deferred mid-blastula transition (MBT) events. In contrast, deleting S phase after the MBT in cycle 15 did not dramatically affect mitotic timing, which appears to retain its dependence on developmentally programmed zygotic transcription. We conclude that normal S phase and replication checkpoint activities are important timers of the undisturbed cell cycle before, but not after, the MBT.

scholarly journals Suppressors of Cdc25p Overexpression Identify Two Pathways That Influence the G2/M Checkpoint in Fission Yeast

Genetics ◽  
1998 ◽  
Vol 150 (4) ◽  
pp. 1361-1375
Author(s):  
Kristi Chrispell Forbes ◽  
Timothy Humphrey ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Gene Encoding ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Dead Box ◽  
New Genes ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
New Gene

Abstract Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25+), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3+). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1+ and the two Schizosaccharomyces pombe 14-3-3 genes, rad24+ and rad25+, which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Δ mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37° and that cds1Δ rad24Δ mutants, like cds1Δ chk1Δ mutants, are entirely checkpoint deficient at 29°. These results suggest that chk1+ and rad24+ may function redundantly with cds1+ in the checkpoint response to unreplicated DNA.

scholarly journals H2A.Z Functions To Regulate Progression through the Cell Cycle

2006 ◽  
Vol 26 (2) ◽  
pp. 489-501 ◽  
Author(s):  
Namrita Dhillon ◽  
Masaya Oki ◽  
Shawn J. Szyjka ◽  
Oscar M. Aparicio ◽  
Rohinton T. Kamakaka
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Histone H2a ◽  
Cycle Progression ◽  
Checkpoint Pathway ◽  
Cyclin Genes

ABSTRACT Histone H2A variants are highly conserved proteins found ubiquitously in nature and thought to perform specialized functions in the cell. Studies in yeast on the histone H2A variant H2A.Z have shown a role for this protein in transcription as well as chromosome segregation. Our studies have focused on understanding the role of H2A.Z during cell cycle progression. We found that htz1Δ cells were delayed in DNA replication and progression through the cell cycle. Furthermore, cells lacking H2A.Z required the S-phase checkpoint pathway for survival. We also found that H2A.Z localized to the promoters of cyclin genes, and cells lacking H2A.Z were delayed in the induction of these cyclin genes. Several different models are proposed to explain these observations.

scholarly journals DNA Replication Checkpoint Control Mediated by the Spindle Checkpoint Protein Mad2p in Fission Yeast

2004 ◽  
Vol 279 (45) ◽  
pp. 47372-47378 ◽  
Author(s):  
Izumi Sugimoto ◽  
Hiroshi Murakami ◽  
Yuko Tonami ◽  
Akihiko Moriyama ◽  
Makoto Nakanishi
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Spindle Checkpoint ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Checkpoint Protein ◽  
Dna Replication Checkpoint

scholarly journals Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

2001 ◽  
Vol 12 (5) ◽  
pp. 1257-1274 ◽  
Author(s):  
Tadayuki Takeda ◽  
Keiko Ogino ◽  
Kazuo Tatebayashi ◽  
Hideo Ikeda ◽  
Ken-ichi Arai ◽  
...  
Keyword(s):  
Dna Replication ◽  
Synthetic Lethality ◽  
S Phase ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Origin Firing ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Pathway ◽  
Nuclear Structures

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect inhsk1-89 is indicated by accumulation ofcut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling.hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those ofrad21-K1 and are significantly enhanced in ahsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

scholarly journals Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression.

1994 ◽  
Vol 125 (4) ◽  
pp. 705-719 ◽  
Author(s):  
S Kornbluth ◽  
M Dasso ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Structure ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
A Cell ◽  
Xenopus Egg Extracts

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP-bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.

scholarly journals Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Urvi Thacker ◽  
Tekle Pauzaite ◽  
James Tollitt ◽  
Maria Twardowska ◽  
Charlotte Harrison ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Zinc Finger Protein ◽  
S Phase ◽  
Cycle Progression ◽  
Inactive X Chromosome ◽  
Functional Characterisation ◽  
Interaction Partners

Abstract CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action.

Sign in / Sign up

Export Citation Format

Share Document