Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Intra-S-phase checkpoint activation by direct CDK2 inhibition.

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.

SUBMITTER: Zhu Y

PROVIDER: S-EPMC434231 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Intra-S-phase checkpoint activation by direct CDK2 inhibition.

Zhu Yonghong Y Alvarez Carmen C Doll Ronald R Kurata Hirokazu H Schebye Xiao Min XM Parry David D Lees Emma E

Molecular and cellular biology 20040701 14

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 ...[more]

PMID: 15226429

Similar Datasets

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe.

Project description:Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.

| S-EPMC6369309 | biostudies-literature

Cdk2 strengthens the intra-S checkpoint and counteracts cell cycle exit induced by DNA damage.

Project description:Although cyclin-dependent kinase 2 (Cdk2) controls the G1/S transition and promotes DNA replication, it is dispensable for cell cycle progression due to redundancy with Cdk1. Yet Cdk2 also has non-redundant functions that can be revealed in certain genetic backgrounds and it was reported to promote the G2/M DNA damage response checkpoint in TP53 (p53)-deficient cancer cells. However, in p53-proficient cells subjected to DNA damage, Cdk2 is inactivated by the CDK inhibitor p21. We therefore investigated whether Cdk2 differentially affects checkpoint responses in p53-proficient and deficient cell lines. We show that, independently of p53 status, Cdk2 stimulates the ATR/Chk1 pathway and is required for an efficient DNA replication checkpoint response. In contrast, Cdk2 is not required for a sustained DNA damage response and G2 arrest. Rather, eliminating Cdk2 delays S/G2 progression after DNA damage and accelerates appearance of early markers of cell cycle exit. Notably, Cdk2 knockdown leads to down-regulation of Cdk6, which we show is a non-redundant pRb kinase whose elimination compromises cell cycle progression. Our data reinforce the notion that Cdk2 is a key p21 target in the DNA damage response whose inactivation promotes exit from the cell cycle in G2.

| S-EPMC5647392 | biostudies-literature

Replication fork integrity and intra-S phase checkpoint suppress gene amplification.

Project description:Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs.

| S-EPMC4357702 | biostudies-literature

NAD<sup>+</sup> bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells.

Project description:Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

| S-EPMC8600031 | biostudies-literature

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing.

Project description:Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53-/-:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53-/- mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.

| S-EPMC3902934 | biostudies-literature

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles.

Project description:Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.

| S-EPMC548659 | biostudies-literature

Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint.

Project description:Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.

| S-EPMC3268413 | biostudies-literature

"DNA Binding Region" of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint.

Project description:The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (?DBR) by using the TALEN method. The ?DBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ?DBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ?DBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ?DBR cells. The ?DBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ?DBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress.

| S-EPMC4737671 | biostudies-other

Nitrosative stress suppresses checkpoint activation after DNA synthesis inhibition.

Project description:DNA synthesis is promoted by the dephosphorylation and activation of cyclin-dependent kinase 2 (Cdk2) complexes by Cdc25A. Nitrosative stress suppresses Cdk2 dephosphorylation by Cdc25A in vitro and inhibits Cdc25A protein translation in cells, but the effects on S-phase progression remain unexamined. Herein we report that nitrosative stress catalyzed by inducible nitric oxide (*NO) synthase (iNOS) or the chemical nitrosant S-nitrosocysteine ethyl ester (SNCEE) rapidly inhibited DNA synthesis concomitant with Cdc25A loss. Surprisingly, this inhibition of DNA synthesis was refractory to ectopic expression of Cdc25A or a Cdc25-independent Cdk2 mutant. Nitrosative stress inhibited DNA synthesis without activating checkpoint signaling, thus distinguishing it from S-phase arrest mediated by other reactive *NO-derived species. The apparent lack of checkpoint activation was due to an active suppression because accumulation of pSer345-Chk1, pThr68-Chk2 and gammaH2AX was inhibited by nitrosative stress in cells exposed to DNA damage or replication inhibitors. We speculate that failure to activate the S-phase checkpoint in precancerous cells undergoing nitrosative stress may elevate the risk of transmitting damaged genomes to daughter cells upon cell cycle reentry.

| S-EPMC2752830 | biostudies-literature

SIRT1 deacetylates TopBP1 and modulates intra-S-phase checkpoint and DNA replication origin firing.

Project description:SIRT1, the mammalian homolog of yeast Sir2, is a founding member of a family of 7 protein and histone deacetylases that are involved in numerous biological functions. Previous studies revealed that SIRT1 deficiency results in genome instability, which eventually leads to cancer formation, yet the underlying mechanism is unclear. To investigate this, we conducted a proteomics study and found that SIRT1 interacted with many proteins involved in replication fork protection and origin firing. We demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. Mechanistically, SIRT1 deacetylates and affects the activity of TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Our study demonstrated that ectopic over-expression of the deacetylated form of TopBP1 in SIRT1 mutant cells repressed replication origin firing, while the acetylated form of TopBP1 lost this function. Thus, SIRT1 acts upstream of TopBP1 and plays an essential role in maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint.

| S-EPMC4261203 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data