scholarly journals Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles

1997 ◽  
Vol 139 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Tin Tin Su ◽  
Patrick H. O'Farrell
Keyword(s):  
Cell Cycle ◽  
Cyclin E ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Chromatin Interaction ◽  
G1 Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycles ◽  
Mcm Proteins ◽  
Replication Factors

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM–chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM–chromatin association.

scholarly journals Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

2011 ◽  
Vol 192 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Marjorie A. Kuipers ◽  
Timothy J. Stasevich ◽  
Takayo Sasaki ◽  
Korey A. Wilson ◽  
Kristin L. Hazelwood ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Replication Fork ◽  
S Phase ◽  
G1 Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycles ◽  
Replication Arrest ◽  
Minichromosome Maintenance Protein ◽  
Mcm Proteins

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.

scholarly journals Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles

1998 ◽  
Vol 140 (3) ◽  
pp. 451-460 ◽  
Author(s):  
Tin Tin Su ◽  
Patrick H. O'Farrell
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Mitotic Cell ◽  
Chromosome Association ◽  
Minichromosome Maintenance ◽  
Chromosome Duplication ◽  
Chromosome Associations ◽  
Mcm Proteins ◽  
Polytene Nuclei ◽  
Replication Factors

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

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.

Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs

1992 ◽  
Vol 102 (2) ◽  
pp. 285-297 ◽  
Author(s):  
D. Leiss ◽  
M.A. Felix ◽  
E. Karsenti
Keyword(s):  
Cell Cycle ◽  
Ribosomal Proteins ◽  
Cell Cycle Progression ◽  
Gradient Centrifugation ◽  
Preliminary Evidence ◽  
Embryonic Cell ◽  
Cytoskeletal Proteins ◽  
Cyclin B ◽  
Post Translational Modifications ◽  
Egg Extracts

Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.

scholarly journals Drosophila Minichromosome Maintenance 6 Is Required for Chorion Gene Amplification and Genomic Replication

2002 ◽  
Vol 13 (2) ◽  
pp. 607-620 ◽  
Author(s):  
Gina Schwed ◽  
Noah May ◽  
Yana Pechersky ◽  
Brian R. Calvi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Genetic Dissection ◽  
Minichromosome Maintenance ◽  
Multiple Origins ◽  
Origin Licensing ◽  
Mcm Complex ◽  
Mcm Proteins ◽  
Prereplicative Complex ◽  
Cycle Regulation

Duplication of the eukaryotic genome initiates from multiple origins of DNA replication whose activity is coordinated with the cell cycle. We have been studying the origins of DNA replication that control amplification of eggshell (chorion) genes duringDrosophila oogenesis. Mutation of genes required for amplification results in a thin eggshell phenotype, allowing a genetic dissection of origin regulation. Herein, we show that one mutation corresponds to a subunit of the minichromosome maintenance (MCM) complex of proteins, MCM6. The binding of the MCM complex to origins in G1 as part of a prereplicative complex is critical for the cell cycle regulation of origin licensing. We find that MCM6 associates with other MCM subunits during amplification. These results suggest that chorion origins are bound by an amplification complex that contains MCM proteins and therefore resembles the prereplicative complex. Lethal alleles of MCM6 reveal it is essential for mitotic cycles and endocycles, and suggest that its function is mediated by ATP. We discuss the implications of these findings for the role of MCMs in the coordination of DNA replication during the cell cycle.

scholarly journals Evolution of CDK1 paralog specializations in a lineage with fast developing planktonic embryos

2019 ◽  
Author(s):  
Xiaofei Ma ◽  
Jan Inge Øvrebø ◽  
Eric M Thompson
Keyword(s):  
General Rule ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Gene Duplications ◽  
Structural Elements ◽  
Oikopleura Dioica ◽  
Cell Cycles ◽  
Protein Levels ◽  
Binding Interface ◽  
M Phase

AbstractThe active site of the essential, eukaryotic CDK1 kinase is generated by core structural elements, among which the PSTAIRE motif in the critical αC-helix, is universally conserved in metazoans. The CDK2 kinase, sharing the PSTAIRE, arose early in metazoan evolution and permitted subdivision of tasks along the S-M-phase axis. The marine chordate, Oikopleura dioica, is the only metazoan known to possess more than a single CDK1 ortholog, and all of its 5 paralogs show sequence divergences in the PSTAIRE. Through assessing CDK1 gene duplications in the appendicularian lineage, we show that the CDK1 activation loop substrate binding platform, ATP entrance site, hinge region, and main Cyclin binding interface, have all diversified under positive selection. Three of the 5 CDK1 paralogs are required for embryonic divisions and knockdown phenotypes illustrate further subdivision of functions along the S-M-phase axis. In parallel to CDK1 gene duplications, there has also been amplification in the Cyclin B complement. Among these, the CDK1d:Cyclin Ba pairing is required for oogenic meiosis and early embryogenesis and shows evidence of coevolution of an exclusive interaction. In an intriguing twist on the general rule that Cyclin B oscillations on a background of stable CDK1 levels regulate M-phase MPF activity, it is CDK1d protein levels that oscillate, rather than Cyclin Ba levels, to drive rapid, early embryonic cell cycles. Strikingly, the modified PSTAIRE of odCDK1d shows convergence over great evolutionary distance with plant CDKB, and in both O. dioica, and plants, these variants exhibit increased specialization to M-phase.

scholarly journals Novel localization and possible functions of cyclin E in early sea urchin development

2002 ◽  
Vol 115 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Bradley J. Schnackenberg ◽  
William F. Marzluff
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Sea Urchin ◽  
Kinase Activity ◽  
Cyclin E ◽  
Sperm Head ◽  
Mitotic Spindles ◽  
Paternal Genome ◽  
Cell Cycles ◽  
Cdk2 Activity

In somatic cells, cyclin E-cdk2 activity oscillates during the cell cycle and is required for the regulation of the G1/S transition. Cyclin E and its associated kinase activity remain constant throughout early sea urchin embryogenesis, consistent with reports from studies using several other embryonic systems. Here we have expanded these studies and show that cyclin E rapidly and selectively enters the sperm head after fertilization and remains concentrated in the male pronucleus until pronuclear fusion, at which time it disperses throughout the zygotic nucleus. We also show that cyclin E is not concentrated at the centrosomes but is associated with condensed chromosomes throughout mitosis for at least the first four cell cycles. Isolated mitotic spindles are enriched for cyclin E and cdk2, which are localized to the chromosomes. The chromosomal cyclin E is associated with active kinase during mitosis. We propose that cyclin E may play a role in the remodeling of the sperm head and re-licensing of the paternal genome after fertilization. Furthermore, cyclin E does not need to be degraded or dissociated from the chromosomes during mitosis; instead, it may be required on chromosomes during mitosis to immediately initiate the next round of DNA replication.

scholarly journals MaternalXenopusCdk2-Cyclin E Complexes Function during Meiotic and Early Embryonic Cell Cycles That Lack a G1Phase

1995 ◽  
Vol 270 (12) ◽  
pp. 6843-6855 ◽  
Author(s):  
Rachel E. Rempel ◽  
Susan B. Sleight ◽  
James L. Maller
Keyword(s):  
Cyclin E ◽  
Embryonic Cell ◽  
Cell Cycles

scholarly journals A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

1997 ◽  
Vol 137 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Xuequn Helen Hua ◽  
Hong Yan ◽  
John Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cyclin E ◽  
Embryonic Cell ◽  
S Phase ◽  
Negative Regulation ◽  
Sperm Chromatin ◽  
High Concentration ◽  
Low Concentrations ◽  
Embryonic Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.

An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3985-3987 ◽  
Author(s):  
Mu-Shui Dai ◽  
Charlie R. Mantel ◽  
Zhen-Biao Xia ◽  
Hal E. Broxmeyer ◽  
Li Lu
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
S Phase ◽  
Cyclin D3 ◽  
G1 Phase ◽  
Cyclin Dependent Kinase ◽  
Terminal Erythroid Differentiation

The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34+cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)–derived erythroblasts from a 7-day culture of CD34+ cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.

Sign in / Sign up

Export Citation Format

Share Document