scholarly journals Cig2, a B-type cyclin, promotes the onset of S in Schizosaccharomyces pombe.

1996 ◽  
Vol 16 (4) ◽  
pp. 1527-1533 ◽  
Author(s):  
O Mondesert ◽  
C H McGowan ◽  
P Russell
Keyword(s):  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Double Mutant ◽  
Kinase Activity ◽  
Nitrogen Starvation ◽  
Gradual Increase ◽  
S Phase ◽  
G1 Arrest ◽  
Cyclin Dependent Kinases ◽  
M Phase

Cdc2, a catalytic subunit of cyclin-dependent kinases, is required for both the G1-to-S and G2-to-M transitions in the fission yeast Schizosaccharomyces pombe. Cdc13, a B-type cyclin, is required for the M-phase induction function of Cd2. Two additional B-type cyclins, Cig1 and Cig2, have been identified in S. pombe, but none of the B-type cyclins are individually required for the onset of S. We report that Cdc13 is important for DNA replication in a strain lacking Cig2. Unlike deltacdc13 cells, double-mutant deltacdc13 deltacig2 cells are defective in undergoing multiple rounds of DNA replication. The conclusion that Cig2 promotes S is further supported by the finding that Cig2 protein and Cig2-associated kinase activity appear soon after the completion of M and peak during S, as well as the observation that S is delayed in deltacig2 cells as they recover from a G1 arrest induced by nitrogen starvation. These studies indicate that Cig2 is the primary S-phase-promoting cyclin in S. pombe but that Cdc13 can effectively substitute for Cig2 in deltacig2 cells. These observations also suggest that the gradual increase in the activity of Cdc2-Cdc13 kinase can be sufficient for the correct temporal ordering of S and M phases in deltacig2 cells.

The anaphase-promoting complex is required in G1 arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S-phase

1997 ◽  
Vol 110 (13) ◽  
pp. 1523-1531 ◽  
Author(s):  
S. Irniger ◽  
K. Nasmyth
Keyword(s):  
Dna Replication ◽  
Physiological Role ◽  
S Phase ◽  
Yeast Cells ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Anaphase Promoting Complex ◽  
Cyclin Dependent Kinases ◽  
Initiation Of Dna Replication ◽  
Mutant Cells

Inactivation of B-type cyclin dependent kinases due to ubiquitin-mediated cyclin proteolysis is necessary for the exit from mitosis. In Saccharomyces cerevisiae, proteolysis is initiated at the onset of anaphase and remains active until Cln1 and Cln2 cyclins appear in late G1 of the subsequent cell cycle. A large particle called the anaphase-promoting complex (APC) which is composed of the TPR proteins Cdc16p/Cdc23p/Cdc27p and other proteins is required for B-type cyclin ubiquitination in both anaphase and during G1 phase. The APC has an essential role for the separation of sister chromatids and for the exit from mitosis, but until now it was unclear whether the persistence of APC activity throughout G1 had any physiological role. We show here that the APC is needed in G1 arrested cells to inhibit premature appearance of B-type cyclins and to prevent unscheduled initiation of DNA replication. When pheromone arrested cells of cdc16 and cdc23 mutants were shifted to the restrictive temperature, they underwent DNA replication in the presence of pheromone. DNA replication also occurred in a G1 arrest induced by G1 cyclin (Cln) depletion, indicating that mutant cells with a defective APC initiate DNA replication without the Cln G1 cyclins, which are normally needed for the onset of S-phase. Degradation of Clb2p, Clb3p and Clb5p depends on the APC. This suggests that accumulation of any one of the six B-type cyclin proteins could account for the precocious replication of cdc16 and cdc23 mutants.

Active cyclin B-cdc2 kinase does not inhibit DNA replication and cannot drive prematurely fertilized sea urchin eggs into mitosis

1995 ◽  
Vol 108 (7) ◽  
pp. 2693-2703 ◽  
Author(s):  
A.M. Geneviere-Garrigues ◽  
A. Barakat ◽  
M. Doree ◽  
J.L. Moreau ◽  
A. Picard
Keyword(s):  
Dna Replication ◽  
Sea Urchin ◽  
S Phase ◽  
Cyclin B ◽  
G1 Arrest ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
M Phase ◽  
Set Up ◽  
The One

Feedback mechanisms preventing M phase occurrence before S phase completion are assumed to depend on inhibition of cyclin B-cdc2 kinase activation by unreplicated DNA. In sea urchin, fertilization stimulates protein synthesis and releases eggs from G1 arrest. We found that in the one-cell sea urchin embryo cyclin B-cdc2 kinase undergoes partial activation before S phase, reaching in S phase a level that is sufficient for G2-M phase transition. S phase entry is not inhibited by this level of cyclin B-dependent kinase activity. Inhibition of DNA replication by aphidicolin suppresses nuclear envelope breakdown, yet it does not prevent the microtubule array from being converted from its interphasic to its mitotic state. Moreover, mitotic cytoplasmic events occur at the same time in control and aphidicolin-treated embryos. Thus unreplicated DNA only prevents mitotic nuclear, not cytoplasmic, events from occurring prematurely. These results together show that the inhibition of cyclin B-cdc2 kinase activation is probably not the only mechanism that prevents mitotic nuclear events from occurring as long as DNA replication has not been completed. In contrast, cytoplasmic mitotic events seem to be controlled by a timing mechanism independent of DNA replication, set up at fertilization, that prevents premature opening of a window for mitotic events.

scholarly journals Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

1996 ◽  
Vol 16 (1) ◽  
pp. 86-93 ◽  
Author(s):  
R Kovelman ◽  
P Russell
Keyword(s):  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Replication Checkpoint ◽  
Activation Assay ◽  
M Phase ◽  
Dna Replication Checkpoint ◽  
High Level ◽  
Tyrosyl Phosphorylation ◽  
In Cells

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.

A checkpoint that monitors cytokinesis in Schizosaccharomyces pombe

2000 ◽  
Vol 113 (7) ◽  
pp. 1223-1230 ◽  
Author(s):  
J. Liu ◽  
H. Wang ◽  
M.K. Balasubramanian
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mutant Allele ◽  
Significant Proportion ◽  
S Phase ◽  
Genetic Screens ◽  
Beta Glucan ◽  
Binucleate Cells ◽  
Number Of Genes ◽  
M Phase ◽  
Ring Constriction

Cell division in Schizosaccharomyces pombe is achieved through the use of a medially positioned actomyosin ring. A division septum is formed centripetally, concomitant with actomyosin ring constriction. Genetic screens have identified mutations in a number of genes that affect actomyosin ring or septum assembly. These cytokinesis-defective mutants, however, undergo multiple S and M phases and die as elongated cells with multiple nuclei. Recently, we have shown that a mutant allele of the S. pombe drc1(+)/cps1(+) gene, which encodes a 1,3-(beta)-glucan synthase subunit, is defective in cytokinesis but displays a novel phenotype. drc1-191/cps1-191 cells are capable of assembling actomyosin rings and completing mitosis, but are incapable of assembling the division septum, causing them to arrest as binucleate cells with a stable actomyosin ring. Each nucleus in arrested cps1-191 cells is able to undergo S phase but these G(2) nuclei are significantly delayed for entry into the M phase. In this study we have investigated the mechanism that causes cps1-191 to block with two G(2) nuclei. We show that the inability of cps1-191 mutants to proceed through multiple mitotic cycles is not related to a defect in cell growth. Rather, the failure to complete some aspect of cytokinesis may prevent the G(2)/M transition of the two interphase-G(2) nuclei. The G(2)/M transition defect of cps1-191 mutants is suppressed by a mutation in the wee1 gene and also by the dominant cdc2 allele cdc2-1w, but not the cdc2-3w allele. Transient depolymerization of all F-actin structures also allowed a significant proportion of the cps1-191 cells to undergo a second round of mitosis. We conclude that an F-actin and Wee1p dependent checkpoint blocks G(2)/M transition until previous cytokinesis is completed.

scholarly journals Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.

1993 ◽  
Vol 123 (6) ◽  
pp. 1321-1331 ◽  
Author(s):  
Y Kubota ◽  
H Takisawa
Keyword(s):  
Dna Replication ◽  
Specific Activity ◽  
S Phase ◽  
Egg Cell ◽  
Sperm Dna ◽  
Before And After ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.

scholarly journals An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication

2019 ◽  
Vol 116 (27) ◽  
pp. 13374-13383 ◽  
Author(s):  
Tatiana N. Moiseeva ◽  
Yandong Yin ◽  
Michael J. Calderon ◽  
Chenao Qian ◽  
Sandra Schamus-Haynes ◽  
...  
Keyword(s):  
Dna Replication ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
S Phase ◽  
Dna Lesions ◽  
Dependent Manner ◽  
Kinase Signaling ◽  
Origin Firing ◽  
Atr Kinase ◽  
Chk1 Kinase

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

scholarly journals The dynamics of replication licensing in live Caenorhabditis elegans embryos

2012 ◽  
Vol 196 (2) ◽  
pp. 233-246 ◽  
Author(s):  
Remi Sonneville ◽  
Matthieu Querenet ◽  
Ashley Craig ◽  
Anton Gartner ◽  
J. Julian Blow
Keyword(s):  
Caenorhabditis Elegans ◽  
Dna Replication ◽  
Dna Binding ◽  
Feedback Loop ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Chromosomal Dna ◽  
M Phase ◽  
Prereplicative Complex

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2–7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2–7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP–ORC-1 bound chromatin independently of ORC-2–5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2–7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2–7 complexes. MCM-2–7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2–7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.

scholarly journals The distribution pattern of proliferating cell nuclear antigen in the nuclei of Leishmania donovani

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3748-3757 ◽  
Author(s):  
Devanand Kumar ◽  
Neha Minocha ◽  
Kalpana Rajanala ◽  
Swati Saha
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Systemic Disease ◽  
S Phase ◽  
Nuclear Antigen ◽  
M Phase ◽  
Proliferating Cell

DNA replication in eukaryotes is a highly conserved process marked by the licensing of multiple origins, with pre-replication complex assembly in G1 phase, followed by the onset of replication at these origins in S phase. The two strands replicate by different mechanisms, and DNA synthesis is brought about by the activity of the replicative DNA polymerases Pol δ and Pol ϵ. Proliferating cell nuclear antigen (PCNA) augments the processivity of these polymerases by serving as a DNA sliding clamp protein. This study reports the cloning of PCNA from the protozoan Leishmania donovani, which is the causative agent of the systemic disease visceral leishmaniasis. PCNA was demonstrated to be robustly expressed in actively proliferating L. donovani promastigotes. We found that the protein was present primarily in the nucleus throughout the cell cycle, and it was found in both proliferating procyclic and metacyclic promastigotes. However, levels of expression of PCNA varied through cell cycle progression, with maximum expression evident in G1 and S phases. The subnuclear pattern of expression of PCNA differed in different stages of the cell cycle; it formed distinct subnuclear foci in S phase, while it was distributed in a more diffuse pattern in G2/M phase and post-mitotic phase cells. These subnuclear foci are the sites of active DNA replication, suggesting that replication factories exist in Leishmania, as they do in higher eukaryotes, thus opening avenues for investigating other Leishmania proteins that are involved in DNA replication as part of these replication factories.

scholarly journals Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content.

1996 ◽  
Vol 7 (2) ◽  
pp. 209-223 ◽  
Author(s):  
N S Datta ◽  
J L Williams ◽  
J Caldwell ◽  
A M Curry ◽  
E K Ashcraft ◽  
...  
Keyword(s):  
Dna Content ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
Cyclin B1 ◽  
S Phase ◽  
H1 Histone ◽  
Protein Levels ◽  
M Phase ◽  
Hel Cells ◽  
Histone Kinase

The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diester-induced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdk1/cyclin B1 complex: a marked reduction in cdk1 protein levels, and an elevated and sustained expression of cyclin B1. Endomitotic cells thus lack cdk1/cyclin B1-associated H1-histone kinase activity. Constitutive over-expression of cdk1 in endomitotic cells failed to re-initiate normal mitotic events even though cdk1 was present in a 10-fold excess. This was due to an inability of cyclin-B1 to physically associate with cdk1. Nonetheless, endomitotic cyclin B1 possesses immunoprecipitable H1-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdk1 and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control.

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