scholarly journals A Genetic Screen for Suppressors and Enhancers of the Drosophila PAN GU Cell Cycle Kinase Identifies Cyclin B as a Target

Genetics ◽  
2001 ◽  
Vol 158 (4) ◽  
pp. 1545-1556 ◽  
Author(s):  
Laura A Lee ◽  
Lisa K Elfring ◽  
Giovanni Bosco ◽  
Terry L Orr-Weaver
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Genetic Screen ◽  
S Phase ◽  
Cyclin B ◽  
Cell Cycles ◽  
Protein Levels ◽  
Drosophila Embryogenesis ◽  
Giant Nuclei ◽  
Cell Cycle Kinase

Abstract The early cell cycles of Drosophila embryogenesis involve rapid oscillations between S phase and mitosis. These unique S-M cycles are driven by maternal stockpiles of components necessary for DNA replication and mitosis. Three genes, pan gu (png), plutonium (plu), and giant nuclei (gnu) are required to control the cell cycle specifically at the onset of Drosophila development by inhibiting DNA replication and promoting mitosis. PNG is a protein kinase that is in a complex with PLU. We employed a sensitized png mutant phenotype to screen for genes that when reduced in dosage would dominantly suppress or enhance png. We screened deficiencies covering over 50% of the autosomes and identified both enhancers and suppressors. Mutations in eIF-5A and PP1 87B dominantly suppress png. Cyclin B was shown to be a key PNG target. Mutations in cyclin B dominantly enhance png, whereas png is suppressed by cyclin B overexpression. Suppression occurs via restoration of Cyclin B protein levels that are decreased in png mutants. The plu and gnu phenotypes are also suppressed by cyclin B overexpression. These studies demonstrate that a crucial function of PNG in controlling the cell cycle is to permit the accumulation of adequate levels of Cyclin B protein.

scholarly journals A Genetic Screen for Suppressors and Enhancers of the Drosophila Cdk1-Cyclin B Identifies Maternal Factors That Regulate Microtubule and Microfilament Stability

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1179-1195 ◽  
Author(s):  
Jun-Yuan Ji ◽  
Marjan Haghnia ◽  
Cory Trusty ◽  
Lawrence S B Goldstein ◽  
Gerold Schubiger
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Genetic Screen ◽  
Cyclin B ◽  
Gene Products ◽  
Nuclear Movement ◽  
Cell Cycles ◽  
Major Mechanism ◽  
Cytoskeletal Dynamics ◽  
Chromosome Bridges

Abstract Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB embryos, we observed patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, we performed a dosage-sensitive genetic screen to identify maternal proteins involved in this process. We identified 10 suppressors classified into three groups: (1) gene products regulating Cdk1 activities, cdk1 and cyclin A; (2) gene products interacting with both microtubules and microfilaments, Actin-related protein 87C; and (3) gene products interacting with microfilaments, chickadee, diaphanous, Cdc42, quail, spaghetti-squash, zipper, and scrambled. Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. Our results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. Our observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage.

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 Rigosertib and Cholangiocarcinoma: A Cell Cycle Affair

2021 ◽  
Vol 23 (1) ◽  
pp. 213
Author(s):  
Alessio Malacrida ◽  
Guido Cavaletti ◽  
Mariarosaria Miloso
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Kinase Inhibitor ◽  
Therapeutic Option ◽  
Cyclin B ◽  
Cell Cycles ◽  
Protein Levels ◽  
M Phase ◽  
Blotting Technique ◽  
A Cell

Rigosertib is multi-kinase inhibitor that could represent an interesting therapeutic option for non-resectable patients with cholangiocarcinoma, a very aggressive hepatic cancer with limited effective treatments. The Western blotting technique was used to evaluate alterations in the expression of proteins involved in the regulation of the cell cycle of cholangiocarcinoma EGI-1 cells. Our results show an increase in EMI1 and Cyclin B protein levels after Rigosertib treatment. Moreover, the phosphorylation of CDK1 is significantly reduced by Rigosertib, while PLK1 expression increased after 24 h of treatment and decreased after 48 h. Finally, we evaluated the role of p53. Its levels increase after Rig treatment, and, as shown in the cell viability experiment with the p53 inhibitor Pifithrin, its activity is necessary for the effects of Rigosertib against the cell viability of EGI-1 cells. In conclusion, we hypothesized the mechanism of the action of Rigosertib against cholangiocarcinoma EGI-1 cells, highlighting the importance of proteins involved in the regulation of cell cycles. The CDK1-Cyclin B complex and p53 play an important role, explaining the Block in the G2/M phase of the cell cycle and the effect on cell viability

Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles

1992 ◽  
Vol 102 (1) ◽  
pp. 63-69 ◽  
Author(s):  
M. Leibovici ◽  
G. Monod ◽  
J. Geraudie ◽  
R. Bravo ◽  
M. Mechali
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Xenopus Laevis ◽  
Early Development ◽  
Nuclear Periphery ◽  
S Phase ◽  
Nuclear Antigen ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Cyclical Pattern

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.

scholarly journals Investigation of the interplay between SKP2, CDT1 and Geminin in cancer cells

2015 ◽  
Author(s):  
Andrea Ballabeni ◽  
Raffaella Zamponi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cancer Cells ◽  
Ubiquitin Ligase ◽  
S Phase ◽  
Physical Interaction ◽  
Protein Levels ◽  
Ubiquitin Ligase Complex ◽  
G2 Phase ◽  
Scf Ubiquitin Ligase

Geminin has a dual role in the regulation of DNA replication: it inhibits replication factor CDT1 activity during the G2 phase of the cell cycle and promotes its accumulation at the G2/M transition. In this way Geminin prevents DNA re-replication during G2 phase and ensures that DNA replication is efficiently executed in the next S phase. CDT1 was shown to associate with SKP2, the substrate recognition factor of the SCF ubiquitin ligase complex. We investigated the interplay between these three proteins in cancer cell lines. We show that Geminin, CDT1 and SKP2 could possibly form a complex and propose the putative regions of CDT1 and Geminin involved in the binding. We also show that, despite the physical interaction, SKP2 depletion does not substantially affect CDT1 and Geminin protein levels. Moreover, we show that while Geminin and CDT1 levels fluctuate, SKP2 levels, differently than in normal cells, are almost steady during the cell cycle of the tested cancer cells.

scholarly journals Investigation of the interplay between SKP2, CDT1 and Geminin in cancer cells

2015 ◽  
Author(s):  
Andrea Ballabeni ◽  
Raffaella Zamponi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cancer Cells ◽  
Ubiquitin Ligase ◽  
S Phase ◽  
Physical Interaction ◽  
Protein Levels ◽  
Ubiquitin Ligase Complex ◽  
G2 Phase ◽  
Scf Ubiquitin Ligase

Geminin has a dual role in the regulation of DNA replication: it inhibits replication factor CDT1 activity during the G2 phase of the cell cycle and promotes its accumulation at the G2/M transition. In this way Geminin prevents DNA re-replication during G2 phase and ensures that DNA replication is efficiently executed in the next S phase. CDT1 was shown to associate with SKP2, the substrate recognition factor of the SCF ubiquitin ligase complex. We investigated the interplay between these three proteins in cancer cell lines. We show that Geminin, CDT1 and SKP2 could possibly form a complex and propose the putative regions of CDT1 and Geminin involved in the binding. We also show that, despite the physical interaction, SKP2 depletion does not substantially affect CDT1 and Geminin protein levels. Moreover, we show that while Geminin and CDT1 levels fluctuate, SKP2 levels, differently than in normal cells, are almost steady during the cell cycle of the tested cancer cells.

scholarly journals The Effect of Dia2 Protein Deficiency on the Cell Cycle, Cell Size, and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 97
Author(s):  
Aneliya Ivanova ◽  
Aleksandar Atemin ◽  
Sonya Uzunova ◽  
Georgi Danovski ◽  
Radoslav Aleksandrov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Cell Size ◽  
Ubiquitin Ligase ◽  
Driving Forces ◽  
S Phase ◽  
Cycle Duration ◽  
Cell Cycles ◽  
Cell Cycle Duration

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCFDia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4—an essential DNA replication protein and substrate of Dia2—prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

Cdc6p establishes and maintains a state of replication competence during G1 phase

1997 ◽  
Vol 110 (6) ◽  
pp. 753-763 ◽  
Author(s):  
C.S. Detweiler ◽  
J.J. Li
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Transcriptional Repression ◽  
Prolonged Exposure ◽  
S Phase ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Important Intermediate ◽  
Initiation Of Dna Replication ◽  
And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

scholarly journals The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle.

1991 ◽  
Vol 11 (8) ◽  
pp. 4045-4052 ◽  
Author(s):  
S Garrett ◽  
M M Menold ◽  
J R Broach
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
S Phase ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Dependent Protein

Null mutations in the gene YAK1, which encodes a protein with sequence homology to known protein kinases, suppress the cell cycle arrest phenotype of mutants lacking the cyclic AMP-dependent protein kinase (A kinase). That is, loss of the YAK1 protein specifically compensates for loss of the A kinase. Here, we show that the protein encoded by YAK1 has protein kinase activity. Yak1 kinase activity is low during exponential growth but is induced at least 50-fold by arrest of cells prior to the completion of S phase. Induction is not observed by arrest at stages later in the cell cycle. Depending on the arrest regimen, induction can occur either by an increase in Yak1 protein levels or by an increase in Yak1 specific activity. Finally, an increase in Yak1 protein levels causes growth arrest of cells with attenuated A kinase activity. These results suggest that Yak1 acts in a pathway parallel to that of the A kinase to negatively regulate cell proliferation.

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