A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.     

Centrosomal Chk2 in DNA damage responses and cell cycle progession

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators.     

Centrosomal Chk2 in DNA damage responses and cell cycle progession

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators.Key words: Chk2, centrosome, checkpoint, DNA damage, wild type, kinase-defective     

Requirement of MTA1 in ATR-mediated DNA Damage Checkpoint Function

MTA1 (metastasis-associated protein 1), an integral component of the nucleosome remodeling and deacetylase complex, has recently been implicated in the ionizing radiation-induced DNA damage response. However, whether MTA1 also participates in the UV-induced DNA damage checkpoint pathway remains unknown. In response to UV radiation, ATR (ataxia teleangiectasia- and Rad3-related) is the major kinase activated that orchestrates cell cycle progression with DNA repair machinery by phosphorylating and activating a number of downstream substrates, such as Chk1 (checkpoint kinase 1) and H2AX (histone 2A variant X). Here, we report that UV radiation stabilizes MTA1 in an ATR-dependent manner and increases MTA1 binding to ATR. On the other hand, depletion of MTA1 compromises the ATR-mediated Chk1 activation following UV treatment, accompanied by a marked down-regulation of Chk1 and its interacting partner Claspin, an adaptor protein that is required for the phosphorylation and activation of Chk1 by ATR. Furthermore, MTA1 deficiency decreases the induction of phosphorylated H2AX (referred to as γ-H2AX) and γ-H2AX focus formation after UV treatment. Consequently, depletion of MTA1 results in a defect in the G₂-M checkpoint and increases cellular sensitivity to UV-induced DNA damage. Thus, MTA1 is required for the activation of the ATR-Claspin-Chk1 and ATR-H2AX pathways following UV treatment, and the noted abrogation of the DNA damage checkpoint in the MTA1-depleted cells may be, at least in part, a consequence of dysregulation of the expression of these two pathways. These findings suggest that, in addition to its role in the repair of double strand breaks caused by ionizing radiation, MTA1 also participates in the UV-induced ATR-mediated DNA damage checkpoint pathway.     

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.     

Lethal Errors in Checkpoint Control: Life without Chk1

no abstract available     

Hec1 Contributes to Mitotic Centrosomal Microtubule Growth for Proper Spindle Assembly through Interaction with Hice1

Previous studies have stipulated Hec1 as a conserved kinetochore component critical for mitotic control in part by directly binding to kinetochore fibers of the mitotic spindle and by recruiting spindle assembly checkpoint proteins Mad1 and Mad2. Hec1 has also been reported to localize to centrosomes, but its function there has yet to be elucidated. Here, we show that Hec1 specifically colocalizes with Hice1, a previously characterized centrosomal microtubule-binding protein, at the spindle pole region during mitosis. In addition, the C-terminal region of Hec1 directly binds to the coiled-coil domain 1 of Hice1. Depletion of Hice1 by small interfering RNA (siRNA) reduced levels of Hec1 in the cell, preferentially at centrosomes and spindle pole vicinity. Reduction of de novo microtubule nucleation from mitotic centrosomes can be observed in cells treated with Hec1 or Hice1 siRNA. Consistently, neutralization of Hec1 or Hice1 by specific antibodies impaired microtubule aster formation from purified mitotic centrosomes in vitro. Last, disruption of the Hec1/Hice1 interaction by overexpressing Hice1ΔCoil1, a mutant defective in Hec1 interaction, elicited abnormal spindle morphology often detected in Hec1 and Hice1 deficient cells. Together, the results suggest that Hec1, through cooperation with Hice1, contributes to centrosome-directed microtubule growth to facilitate establishing a proper mitotic spindle.     

The Interaction between Checkpoint Kinase 1 (Chk1) and the Minichromosome Maintenance (MCM) Complex Is Required for DNA Damage-induced Chk1 Phosphorylation

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage.     

Resisting Arrest: Recovery from Checkpoint Arrest Through Dephosphorylation of Chk1 by PP1

The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signalling, and speculate on models for checkpoint maintenance and release.     

Basis for the Checkpoint Signal Specificity That Regulates Chk1 and Cds1 Protein Kinases

Six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are needed to regulate checkpoint protein kinases Chk1 and Cds1 in fission yeast. Chk1 is required to prevent mitosis when DNA is damaged by ionizing radiation (IR), whereas either kinase is sufficient to prevent mitosis when DNA replication is inhibited by hydroxyurea (HU). Checkpoint Rad proteins are required for IR-induced phosphorylation of Chk1 and HU-induced activation of Cds1. IR activates Cds1 only during the DNA synthesis (S) phase, whereas HU induces Chk1 phosphorylation only in cds1 mutants. Here, we investigate the basis of the checkpoint signal specificity of Chk1 phosphorylation and Cds1 activation. We show that IR fails to induce Chk1 phosphorylation in HU-arrested cells. Release from the HU arrest following IR causes substantial Chk1 phosphorylation. These and other data indicate that Cds1 prevents Chk1 phosphorylation in HU-arrested cells, which suggests that Cds1 actively suppresses a repair process that leads to Chk1 phosphorylation. Cds1 becomes more highly concentrated in the nucleus only during the S phase of the cell cycle. This finding correlates with S-phase specificity of IR-induced activation of Cds1. However, constitutive nuclear localization of Cds1 does not enhance IR-induced activation of Cds1. This result suggests that Cds1 activation requires DNA structures or protein activities that are present only during S phase. These findings help to explain how Chk1 and Cds1 respond to different checkpoint signals.     

Chk1 is Essential for Tumor Cell Viability Following Activation of the Replication Checkpoint

Type I interferons (IFNs) are a family of cytokines that exhibit various biological activities. Besides their roles in immune response, IFNs have been known to modulate cell proliferation and to induce apoptosis. Thus, IFNs are used as an anti-tumor agent against certain types of cancer, but it is unclear why many other cancers are not influenced by IFNs. Here, we found that IFN-a2b, a subfamily of IFN-a, enhanced proliferation of HeLa cells, a cell line derived from human cervical cancer. IFN-a2b was rather inhibitory on the growth of other types of cervical cancer cells including those positive for HPV. Among the proliferation- and the apoptosis-related genes, p21^cip1/waf1 (p21) was upregulated by IFN-a2b, whereas p53, p27 or BCL-2 associated X protein (BAX) was not affected. IFN-a2b did not alter promoter activities of p21 but did prolong the decay of p21 mRNA. In contrast, the level of p21 protein was lowered by IFN-a2b, and half-life analysis of p21 protein revealed that IFN-a2b enhances p21 protein instability in HeLa cells. Pretreatment of the cells with MG132, a proteasome inhibitor, abolished the IFN-a2b-mediated p21 degradation, suggesting that IFN-a2b accelerated the ubiquitin-proteasome dependent degradation of p21. Consistent with these results, IFN-a2b increased S-phase cell cycle distribution in HeLa cells. In addition, IFN-a2b liberated the cells from G1-phase arrest by 5-fluorouracil (5-FU) and from G2-phase arrest by paclitaxel. These results provide a novel role of Type I IFNs in cell cycle regulation and may define an importance of individualized IFN-based therapy against specific types of cancer.     

Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.     

Toxic Effect of Silica Nanoparticles on Endothelial Cells through DNA Damage Response via Chk1-Dependent G2/M Checkpoint

Silica nanoparticles have become promising carriers for drug delivery or gene therapy. Endothelial cells could be directly exposed to silica nanoparticles by intravenous administration. However, the underlying toxic effect mechanisms of silica nanoparticles on endothelial cells are still poorly understood. In order to clarify the cytotoxicity of endothelial cells induced by silica nanoparticles and its mechanisms, cellular morphology, cell viability and lactate dehydrogenase (LDH) release were observed in human umbilical vein endothelial cells (HUVECs) as assessing cytotoxicity, resulted in a dose- and time- dependent manner. Silica nanoparticles-induced reactive oxygen species (ROS) generation caused oxidative damage followed by the production of malondialdehyde (MDA) as well as the inhibition of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Both necrosis and apoptosis were increased significantly after 24 h exposure. The mitochondrial membrane potential (MMP) decreased obviously in a dose-dependent manner. The degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM) were markedly aggravated. Silica nanoparticles also induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. In summary, our data indicated that the toxic effect mechanisms of silica nanoparticles on endothelial cells was through DNA damage response (DDR) via Chk1-dependent G2/M checkpoint signaling pathway, suggesting that exposure to silica nanoparticles could be a potential hazards for the development of cardiovascular diseases.     

Cdc25 Inhibited In Vivo and In Vitro by Checkpoint Kinases Cds1 and Chk1

In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G2-M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S-M replication and G2-M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.     

BRCA1 and Chk1 in G2/M Checkpoint: A New Order of Regulation

No Abstract Available

Key Words:

DNA damage, Checkpoints, BRCA1, Chk1