scholarly journals Intra-S-Phase Checkpoint Activation by Direct CDK2 Inhibition

2004 ◽  
Vol 24 (14) ◽  
pp. 6268-6277 ◽  
Author(s):  
Yonghong Zhu ◽  
Carmen Alvarez ◽  
Ronald Doll ◽  
Hirokazu Kurata ◽  
Xiao Min Schebye ◽  
...  
Keyword(s):  
Genomic Stability ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Minichromosome Maintenance ◽  
Checkpoint Activation ◽  
Cdk2 Activity ◽  
A Cell ◽  
Dependent Pathway ◽  
Number Of Cells ◽  
S Phase Checkpoint

ABSTRACT To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.

scholarly journals The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted

2007 ◽  
Vol 179 (4) ◽  
pp. 643-657 ◽  
Author(s):  
Enbo Liu ◽  
Alan Yueh-Luen Lee ◽  
Takuya Chiba ◽  
Erin Olson ◽  
Peiqing Sun ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein A ◽  
S Phase ◽  
Minichromosome Maintenance ◽  
Checkpoint Activation ◽  
Checkpoint Pathway ◽  
Surveillance Mechanism ◽  
Dna Rereplication ◽  
S Phase Checkpoint

DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)–mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.

scholarly journals Divergent S Phase Checkpoint Activation Arising from Prereplicative Complex Deficiency Controls Cell Survival

2009 ◽  
Vol 20 (17) ◽  
pp. 3953-3964 ◽  
Author(s):  
Eric Lau ◽  
Gary G. Chiang ◽  
Robert T. Abraham ◽  
Wei Jiang
Keyword(s):  
Dna Replication ◽  
Cancer Cells ◽  
S Phase ◽  
Checkpoint Control ◽  
Multiple Cancer ◽  
Checkpoint Activation ◽  
Diploid Cells ◽  
Prereplicative Complex ◽  
Stress Damage ◽  
S Phase Checkpoint

The DNA replication machinery plays additional roles in S phase checkpoint control, although the identities of the replication proteins involved in checkpoint activation remain elusive. Here, we report that depletion of the prereplicative complex (pre-RC) protein Cdc6 causes human nontransformed diploid cells to arrest nonlethally in G1-G1/S and S phase, whereas multiple cancer cell lines undergo G1-G1/S arrest and cell death. These divergent phenotypes are dependent on the activation, or lack thereof, of an ataxia telangiectasia and Rad3-related (ATR)-dependent S phase checkpoint that inhibits replication fork progression. Although pre-RC deficiency induces chromatin structural alterations in both nontransformed and cancer cells that normally lead to ATR checkpoint activation, the sensor mechanisms in cancer cells seem to be compromised such that higher levels of DNA replication stress/damage are required to trigger checkpoint response. Our results suggest that therapy-induced disruption of pre-RC function might exert selective cytotoxic effects on tumor cells in human patients.

scholarly journals Two independent S-phase checkpoints regulate appressorium-mediated plant infection by the rice blast fungusMagnaporthe oryzae

2016 ◽  
Vol 114 (2) ◽  
pp. E237-E244 ◽  
Author(s):  
Míriam Osés-Ruiz ◽  
Wasin Sakulkoo ◽  
George R. Littlejohn ◽  
Magdalena Martin-Urdiroz ◽  
Nicholas J. Talbot
Keyword(s):  
Rice Blast ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Blast Disease ◽  
Actin Dynamics ◽  
Phase Cell ◽  
Physical Force ◽  
Rice Leaf ◽  
Plant Infection ◽  
S Phase Checkpoint

To cause rice blast disease, the fungal pathogenMagnaporthe oryzaedevelops a specialized infection structure called an appressorium. This dome-shaped, melanin-pigmented cell generates enormous turgor and applies physical force to rupture the rice leaf cuticle using a rigid penetration peg. Appressorium-mediated infection requires septin-dependent reorientation of the F-actin cytoskeleton at the base of the infection cell, which organizes polarity determinants necessary for plant cell invasion. Here, we show that plant infection byM. oryzaerequires two independent S-phase cell-cycle checkpoints. Initial formation of appressoria on the rice leaf surface requires an S-phase checkpoint that acts through the DNA damage response (DDR) pathway, involving the Cds1 kinase. By contrast, appressorium repolarization involves a novel, DDR-independent S-phase checkpoint, triggered by appressorium turgor generation and melanization. This second checkpoint specifically regulates septin-dependent, NADPH oxidase-regulated F-actin dynamics to organize the appressorium pore and facilitate entry of the fungus into host tissue.

scholarly journals Snm1B/Apollo mediates replication fork collapse and S Phase checkpoint activation in response to DNA interstrand cross-links

Oncogene ◽  
2008 ◽  
Vol 27 (37) ◽  
pp. 5045-5056 ◽  
Author(s):  
J-B Bae ◽  
S S Mukhopadhyay ◽  
L Liu ◽  
N Zhang ◽  
J Tan ◽  
...  
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Checkpoint Activation ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
S Phase Checkpoint

scholarly journals Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
WooKee Min ◽  
Christopher Bruhn ◽  
Paulius Grigaravicius ◽  
Zhong-Wei Zhou ◽  
Fu Li ◽  
...  
Keyword(s):  
S Phase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Stalled Replication Forks ◽  
S Phase Checkpoint

scholarly journals Rb function is required for E1A-induced S-phase checkpoint activation

2008 ◽  
Vol 15 (9) ◽  
pp. 1440-1449 ◽  
Author(s):  
A Nemajerova ◽  
F Talos ◽  
U M Moll ◽  
O Petrenko
Keyword(s):  
S Phase ◽  
Checkpoint Activation ◽  
S Phase Checkpoint

Enhanced Phosphorylation of Nbs1, a Member of the DNA Repair/Checkpoint Activation Complex Rad50/Mre11/Nbs1, Prolongs Cell Cycle S Phase and Contributes to Drug Resistance in BCR/ABL-Positive Leukemias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2867-2867 ◽  
Author(s):  
Lori Rink ◽  
Tomasz Stoklosa ◽  
Tomasz Skorski
Keyword(s):  
Dna Repair ◽  
Growth Factors ◽  
Dna Synthesis ◽  
Cell Lines ◽  
S Phase ◽  
Leukemia Cells ◽  
Checkpoint Activation ◽  
Normal Cells ◽  
Nuclear Foci ◽  
S Phase Checkpoint

Abstract Nbs1, a member of the DNA repair/checkpoint activation complex Mre11/Rad50/Nbs1, is phosphorylated by ATM in response to the presence of DNA double strand breaks (DSBs) resulting in the activation of the S phase checkpoint. Here we show that the BCR/ABL tyrosine kinase, as well as growth factors (IL-3, GM-CSF), stimulate the expression of Mre11 and Nbs1, but not Rad50. This effect is dependent on the kinase activity of BCR/ABL protecting Mre11 and Nbs1 from caspase-dependent, but not proteasome-dependent degradation. Cells expressing the BCR/ABL kinase are resistant to chemotherapeutic agents, including mitomycin C (MMC). Western analysis showed enhanced phosphorylation of Nbs1 on Serine 343 in MMC-treated BCR/ABL leukemia cells (CML patient cells and leukemic cell lines) in comparison to normal cells in the presence of growth factors. Immunofluoresence studies demonstrated an increase of γ-H2AX nuclear foci (DSBs indicator) and pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. In addition, BCR/ABL-positive cells displayed a higher percentage of colocalization of γ-H2AX and p-Nbs1 implicating the presence of p-Nbs1 at the DSBs. Clonogenic assays performed after down regulation of Nbs1 in BCR/ABL positive cells using siRNA showed increased sensitivity to MMC. Specifically, the expression of the Nbs1-S343A phosphorylation-less mutant also decreased resistance in BCR/ABL cells to MMC. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells, BCR/ABL-transformed cell lines and normal counterparts displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase. Expression of Nbs1-S343A mutant caused a significant decrease in the accumulation of BCR/ABL leukemia cells in S phase after MMC treatment, whereas cells transfected with both the empty construct and wild-type Nbs1 displayed S phase accumulation. Surprisingly, Nbs1-S343A mutant did not affect the ability of normal cells to accumulate in S phase in response to MMC. Altogether, we hypothesize that enhanced phosphorylation of Nbs1 on S343 leads to increased resistance to genotoxic agents in BCR/ABL leukemia cells by prolonging the S phase checkpoint and allowing longer time for the repair of excessive DNA damage.

scholarly journals Cell-Free Degradation of p27kip1, a G1 Cyclin-Dependent Kinase Inhibitor, Is Dependent on CDK2 Activity and the Proteasome

1999 ◽  
Vol 19 (2) ◽  
pp. 1190-1201 ◽  
Author(s):  
Hoang Nguyen ◽  
Diana M. Gitig ◽  
Andrew Koff
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
S Phase ◽  
Cycle Phase ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Cdk2 Activity ◽  
A Cell ◽  
Specific Factors ◽  
Specific Degradation

ABSTRACT Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G1/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.

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