scholarly journals Human Cyclin a Is Required for Mitosis until Mid Prophase

1999 ◽  
Vol 147 (2) ◽  
pp. 295-306 ◽  
Author(s):  
Nobuaki Furuno ◽  
Nicole den Elzen ◽  
Jonathon Pines
Keyword(s):  
Cyclin A ◽  
Time Lapse ◽  
S Phase ◽  
Early Prophase ◽  
Cyclin Dependent Kinase ◽  
Late Prophase ◽  
Daughter Cells ◽  
G2 Phase ◽  
Rate Limiting

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

scholarly journals Cell cycle inertia underlies a bifurcation in cell fates after DNA damage

2021 ◽  
Vol 7 (3) ◽  
pp. eabe3882
Author(s):  
Jenny F. Nathans ◽  
James A. Cornwell ◽  
Marwa M. Afifi ◽  
Debasish Paul ◽  
Steven D. Cappell
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Tracking ◽  
Time Lapse ◽  
S Phase ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Cell Fates ◽  
Damage Response ◽  
Time Lapse Imaging

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

scholarly journals Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sabrina F Mansilla ◽  
Agustina P Bertolin ◽  
Valérie Bergoglio ◽  
Marie-Jeanne Pillaire ◽  
Marina A González Besteiro ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Genomic Stability ◽  
S Phase ◽  
Fragile Sites ◽  
Dna Lesions ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Independent Manner ◽  
Genomic Regions

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

Nucleolus behaviour during the cell cycle of a primitive dinoflagellate eukaryote, Prorocentrum micans Ehr., seen by light microscopy and electron microscopy

1992 ◽  
Vol 102 (3) ◽  
pp. 475-485 ◽  
Author(s):  
MARIE-ODILE SOYER-GOBILLARD ◽  
MARIE-LINE GERAUD
Keyword(s):  
Cell Cycle ◽  
Light Microscopy ◽  
Early Prophase ◽  
Microscopy Observation ◽  
Prorocentrum Micans ◽  
Late Prophase ◽  
Daughter Cells ◽  
Nucleolar Material ◽  
Freeze Fixation ◽  
Higher Eukaryotes

Light-microscopy observation of the dinoflagellate Prorocentrum micans after silver-staining of the argyrophilic proteins of the nucleolar organizing region (Ag-NOR staining) showed the presence of nucleolar material throughout the vegetative cell cycle, and in particular during all the mitotic stages. This contrasts with the case in most higher eukaryotes, in which nucleoli disappear at the end of prophase and are reconstituted in daughter cells during telophase. Electron-microscope (EM) observations after conventional or fast-freeze fixation revealed that during interphase several functional nucleoli with three compartments (NORs, the fibrillogranular and the preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early prophase, when chromosomes are beginning to split, the nucleoli remain functional, whereas in late prophase they contain only a NOR and the granular component, and the chromosomes are surrounded by many protein masses. In early telophase, the nucleolar material coating the chromosomes migrates along with the chromosomes. Nucleologenesis occurs through the formation of prenucleolar bodies around lateral or telomeric nucleofilaments extruding from the chromosomes. Several chromosomes can contribute to the formation of one nucleolus. The behaviour of these ‘persistent nucleoli’ in a closed-nucleus model such as that of the dinoflagellates is discussed with regard to the higher eukaryotes.

scholarly journals LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

1966 ◽  
Vol 31 (3) ◽  
pp. 577-583 ◽  
Author(s):  
J. E. Cummins ◽  
H. P. Rusch
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Nuclear Dna ◽  
S Phase ◽  
Early Prophase ◽  
Late Prophase ◽  
Removal Experiments ◽  
Inhibitor Of Protein Synthesis

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.

scholarly journals Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroaki Shimono ◽  
Atsushi Kaida ◽  
Hisao Homma ◽  
Hitomi Nojima ◽  
Yusuke Onozato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Time Lapse ◽  
S Phase ◽  
G1 Phase ◽  
M Phase ◽  
G2 Phase ◽  
The Difference ◽  
Time Lapse Imaging ◽  
First Time

AbstractIn this study, we examined the fluctuation in radioresponse of HeLa cells during the cell cycle. For this purpose, we used HeLa cells expressing two types of fluorescent ubiquitination-based cell cycle indicators (Fucci), HeLa-Fucci (CA)2 and HeLa-Fucci (SA), and combined this approach with the micronucleus (MN) assay to assess radioresponse. The Fucci system distinguishes cell cycle phases based on the colour of fluorescence and cell morphology under live conditions. Time-lapse imaging allowed us to further identify sub-positions within the G1 and S phases at the time of irradiation by two independent means, and to quantitate the number of MNs by following each cell through M phase until the next G1 phase. Notably, we found that radioresponse was low in late G1 phase, but rapidly increased in early S phase. It then decreased until late S phase and increased in G2 phase. For the first time, we demonstrated the unique fluctuation of radioresponse by the MN assay during the cell cycle in HeLa cells. We discuss the difference between previous clonogenic experiments using M phase-synchronised cell populations and ours, as well as the clinical implications of the present findings.

scholarly journals A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited

2011 ◽  
Vol 366 (1584) ◽  
pp. 3572-3583 ◽  
Author(s):  
Frank Uhlmann ◽  
Céline Bouchoux ◽  
Sandra López-Avilés
Keyword(s):  
Cell Cycle ◽  
Eukaryotic Cell ◽  
Quantitative Model ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Genomic Integrity ◽  
Chromosomal Dna ◽  
Substrate Specificities ◽  
Daughter Cells ◽  
Molecular Events

The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed ‘A quantitative model for cdc2 control of S phase and mitosis in fission yeast’. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications.

scholarly journals Noncatalytic Requirement for Cyclin A-cdk2 in p27 Turnover

2004 ◽  
Vol 24 (13) ◽  
pp. 6058-6066 ◽  
Author(s):  
Xin-Hua Zhu ◽  
Hoang Nguyen ◽  
H. Dorota Halicka ◽  
Frank Traganos ◽  
Andrew Koff
Keyword(s):  
Ubiquitin Ligase ◽  
Cyclin E ◽  
Flow Cytometric Analysis ◽  
E3 Ubiquitin Ligase ◽  
Cyclin A ◽  
Cyclin B1 ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Flow Cytometric

ABSTRACT Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27kip1 is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G1 cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCFskp2 complex.

scholarly journals The role of DDK and Treslin-MTBP in coordinating replication licensing and pre-Initiation Complex formation

2021 ◽  
Author(s):  
Ilaria Volpi ◽  
Peter J. Gillespie ◽  
Gaganmeet Singh Chadha ◽  
J. Julian Blow
Keyword(s):  
Dna Replication ◽  
Complex Formation ◽  
S Phase ◽  
Replication Initiation ◽  
Initiation Complex ◽  
Crucial Step ◽  
Egg Extract ◽  
Initiation Of Dna Replication ◽  
Rate Limiting

AbstractTreslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin-MTBP is rate-limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin-MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin-MTBP with TopBP1 which is a regulated crucial step in pre-Initiation Complex formation. These results suggest how DDK works together with CDKs to regulate Treslin-MTBP and plays a crucial in selecting which origins will undergo initiation.

scholarly journals The role of DDK and Treslin–MTBP in coordinating replication licensing and pre-initiation complex formation

Open Biology ◽  
2021 ◽  
Vol 11 (10) ◽  
Author(s):  
Ilaria Volpi ◽  
Peter J. Gillespie ◽  
Gaganmeet Singh Chadha ◽  
J. Julian Blow
Keyword(s):  
Dna Replication ◽  
Complex Formation ◽  
S Phase ◽  
Replication Initiation ◽  
Initiation Complex ◽  
Crucial Step ◽  
Egg Extract ◽  
Initiation Of Dna Replication ◽  
Rate Limiting

Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin–MTBP is rate limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin–MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin–MTBP with TopBP1 which is a regulated crucial step in pre-initiation complex formation. These results suggest how DDK works together with CDKs to regulate Treslin–MTBP and plays a crucial in selecting which origins will undergo initiation.

scholarly journals A Protein Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 Is Expressed in Trigeminal Ganglionic Neurons of Latently Infected Cattle and Interacts with Cyclin-Dependent Kinase 2 during Productive Infection

1998 ◽  
Vol 72 (10) ◽  
pp. 8133-8142 ◽  
Author(s):  
Yunquan Jiang ◽  
Ashfaque Hossain ◽  
Maria Teresa Winkler ◽  
Todd Holt ◽  
Alan Doster ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Acute Infection ◽  
Cyclin A ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Bovine Herpesvirus 1 ◽  
Cycle Progression ◽  
Latently Infected ◽  
Herpesvirus 1

ABSTRACT Despite productive viral gene expression in the peripheral nervous system during acute infection, the bovine herpesvirus 1 (BHV-1) infection cycle is blocked in sensory ganglionic neurons and consequently latency is established. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. LR gene products inhibit S-phase entry, and binding of the LR protein (LRP) to cyclin A was hypothesized to block cell cycle progression. This study demonstrates LRP is a nuclear protein which is expressed in neurons of latently infected cattle. Affinity chromatography indicated that LRP interacts with cyclin-dependent kinase 2 (cdk2)-cyclin complexes or cdc2-cyclin complexes in transfected human cells or infected bovine cells. After partial purification using three different columns (DEAE-Sepharose, Econo S, and heparin-agarose), LRP was primarily associated with cdk2-cyclin E complexes, an enzyme which is necessary for G1-to-S-phase cell cycle progression. During acute infection of trigeminal ganglia or following dexamethasone-induced reactivation, BHV-1 induces expression of cyclin A in neurons (L. M. Schang, A. Hossain, and C. Jones, J. Virol. 70:3807–3814, 1996). Expression of S-phase regulatory proteins (cyclin A, for example) leads to neuronal apoptosis. Consequently, we hypothesize that interactions between LRP and cell cycle regulatory proteins promote survival of postmitotic neurons during acute infection and/or reactivation.

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