共查询到20条相似文献,搜索用时 15 毫秒
1.
Xiangzi Han Aaron Aslanian Kang Fu Toshiya Tsuji Youwei Zhang 《The Journal of biological chemistry》2014,289(35):24716-24723
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. 相似文献
2.
Jiang K Pereira E Maxfield M Russell B Goudelock DM Sanchez Y 《The Journal of biological chemistry》2003,278(27):25207-25217
Checkpoints are biochemical pathways that provide the cell with mechanisms to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase Chk1 regulates mitotic progression in response to DNA damage and replication interference by blocking the activation of Cdk1/cyclin B. Chk1 is phosphorylated on Ser-317 and Ser-345 following a checkpoint signal, a process that is regulated by Atr, and by the sensor complexes containing Rad17 and Hus1. We show that Chk1 is associated with chromatin in cycling cells and that the chromatin-associated Chk1 is phosphorylated in the absence of exogenous DNA damage. The UV-induced Ser-345-phosphorylated forms of Chk1 that appear minutes after treatment are predominantly associated with chromatin. The Ser-345 site is in a 14-3-3 consensus binding motif and is required for nuclear retention of Chk1 following an hydroxyurea-induced checkpoint signal; nonetheless, Ser-345 or Ser-317 are not required for the chromatin association of Chk1. Hus1, a member of the proliferating cell nuclear antigen-like damage recognition complex plays a role in the phosphorylation of Chk1 on Ser-345, however, Hus1 is not required for phosphorylation on Ser-317 or for Chk1 localization to chromatin. These results indicate that there is more than one step in Chk1 activation and that the regulation of this checkpoint signaling is achieved at least in part through phosphorylation of Ser-345, which serves to localize Chk1 in the nucleus presumably by blocking Crm1-dependent nuclear export. 相似文献
3.
The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase. 相似文献
4.
Qian Wei Junhui Li Ting Liu Xiaomei Tong Xin Ye 《The Journal of biological chemistry》2013,288(27):19715-19725
MCM7 is one of the subunits of the MCM2–7 complex that plays a critical role in DNA replication initiation and cell proliferation of eukaryotic cells. After forming the pre-replication complex (pre-RC) with other components, the MCM2–7 complex is activated by DDK/cyclin-dependent kinase to initiate DNA replication. Each subunit of the MCM2–7 complex functions differently under regulation of various kinases on the specific site, which needs to be investigated in detail. In this study, we demonstrated that MCM7 is a substrate of cyclin E/Cdk2 and can be phosphorylated on Ser-121. We found that the distribution of MCM7-S121A is different from wild-type MCM7 and that the MCM7-S121A mutant is much less efficient to form a pre-RC complex with MCM3/MCM5/cdc45 compared with wild-type MCM7. By using the Tet-On inducible HeLa cell line, we revealed that overexpression of wild-type MCM7 but not MCM7-S121A can block S phase entry, suggesting that an excess of the pre-RC complex may activate the cell cycle checkpoint. Further analysis indicates that the Chk1 pathway is activated in MCM7-overexpressed cells in a p53-dependent manner. We performed experiments with the human normal cell line HL-7702 and also observed that overexpression of MCM7 can cause S phase block through checkpoint activation. In addition, we found that MCM7 could also be phosphorylated by cyclin B/Cdk1 on Ser-121 both in vitro and in vivo. Furthermore, overexpression of MCM7-S121A causes an obvious M phase exit delay, which suggests that phosphorylation of MCM7 on Ser-121 in M phase is very important for a proper mitotic exit. These data suggest that the phosphorylation of MCM7 on Ser-121 by cyclin/Cdks is involved in preventing DNA rereplication as well as in regulation of the mitotic exit. 相似文献
5.
The minichromosome maintenance (MCM) complex, a replicative helicase, is a heterohexamer essential for DNA duplication and genome stability. We identified Schizosaccharomyces pombe mcb1(+) (Mcm-binding protein 1), an apparent orthologue of the human MCM-binding protein that associates with a subset of MCM complex proteins. mcb1(+) is an essential gene. Deletion of mcb1(+) caused cell cycle arrest after several generations with a cdc phenotype and disrupted nuclear structure. Mcb1 is an abundant protein, constitutively present across the cell cycle. It is widely distributed in cytoplasm and nucleoplasm and bound to chromatin. Co-immunoprecipitation suggested that Mcb1 interacts robustly with Mcm3-7 but not Mcm2. Overproduction of Mcb1 disrupted the association of Mcm2 with other MCM proteins, resulting in inhibition of DNA replication, DNA damage, and activation of the checkpoint kinase Chk1. Thus, Mcb1 appears to antagonize the function of MCM helicase. 相似文献
6.
Da-Qiang Li Kazufumi Ohshiro Mudassar N. Khan Rakesh Kumar 《The Journal of biological chemistry》2010,285(26):19802-19812
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. 相似文献
7.
Jean-Marc Brondello Michael N. Boddy Beth Furnari Paul Russell 《Molecular and cellular biology》1999,19(6):4262-4269
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. 相似文献
8.
Michael D. Rainey Brendan Harhen Guan-Nan Wang Paul V. Murphy Corrado Santocanale 《Cell cycle (Georgetown, Tex.)》2013,12(10):1560-1568
Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin’s function in the DNA replication checkpoint. 相似文献
9.
Pankaj Chaudhary Rajendra Sharma Mukesh Sahu Jamboor K. Vishwanatha Sanjay Awasthi Yogesh C. Awasthi 《The Journal of biological chemistry》2013,288(28):20532-20546
4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested. 相似文献
10.
Guoqi Liu Sheré Myers Xiaomi Chen John J. Bissler Richard R. Sinden Michael Leffak 《The Journal of biological chemistry》2012,287(40):33412-33423
DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response. 相似文献
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13.
Rémy Bétous Marie‐Jeanne Pillaire Laura Pierini Siem van der Laan Emma Ohl‐Séguy Caixia Guo Naoko Niimi Petr Grúz Takehiko Nohmi Errol Friedberg Christophe Cazaux Domenico Maiorano Jean‐Sébastien Hoffmann 《The EMBO journal》2013,32(15):2172-2185
Formation of primed single‐stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR‐mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA‐mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y‐family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9‐1‐1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9‐1‐1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells. 相似文献
14.
MCM2-7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2-7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2-7 proteins contributes to the formation of the MCM2-7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2-7 complex. 相似文献
15.
Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation
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The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45‐MCM‐GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK‐dependent manner. Sld3 binds specifically to DDK‐phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho‐MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK‐independent replication. Thus, Sld3 is an essential “reader” of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. 相似文献
16.
Laura A. Lindsey-Boltz ?zdemirhan Ser?in Jun-Hyuk Choi Aziz Sancar 《The Journal of biological chemistry》2009,284(48):33107-33114
ATR (ATM and Rad3-related) initiates a DNA damage signaling pathway in human cells upon DNA damage induced by UV and UV-mimetic agents and in response to inhibition of DNA replication. Genetic data with human cells and in vitro data with Xenopus egg extracts have led to the conclusion that the kinase activity of ATR toward the signal-transducing kinase Chk1 depends on the mediator protein Claspin. Here we have reconstituted a Claspin-mediated checkpoint system with purified human proteins. We find that the ATR-dependent phosphorylation of Chk1, but not p53, is strongly stimulated by Claspin. Similarly, DNA containing bulky base adducts stimulates ATR kinase activity, and Claspin acts synergistically with damaged DNA to increase phosphorylation of Chk1 by ATR. Mutations in putative phosphorylation sites in the Chk1-binding domain of Claspin abolish its ability to mediate ATR phosphorylation of Chk1. We also find that a fragment of Claspin containing the Chk1-binding domain together with a domain conserved in the yeast Mrc1 orthologs of Claspin is sufficient for its mediator activity. This in vitro system recapitulates essential components of the genetically defined ATR-signaling pathway. 相似文献
17.
Regulatory interactions between the checkpoint kinase Chk1 and the proteins of the DNA-dependent protein kinase complex 总被引:3,自引:0,他引:3
Goudelock DM Jiang K Pereira E Russell B Sanchez Y 《The Journal of biological chemistry》2003,278(32):29940-29947
Checkpoints are biochemical pathways that provide cells a mechanism to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase, Chk1, regulates mitotic progression in response to DNA damage by blocking the activation of Cdk1/cyclin B. In this study, we investigate the regulatory interaction between Chk1 and members of the Atm family of kinases and the functional role of the C-terminal non-catalytic domains of Chk1. Chk1 stimulates the kinase activity of DNA-PK (protein kinase) complexes, which leads to increased phosphorylation of p53 on Ser-15 and Ser-37. In addition, Chk1 stimulates DNA-PK-dependent end-joining reactions in vitro. We also show that Chk1 protein complexes bind to single-stranded DNA and DNA ends. These results indicate a connection between components that regulate the checkpoint pathways and DNA-PK complex proteins, which have a role in the repair of double strand breaks. 相似文献
18.
Genotoxic stress activates checkpoint signaling pathways that block cell cycle progression, trigger apoptosis, and regulate DNA repair. Studies in yeast and humans have shown that Rad9, Hus1, Rad1, and Rad17 play key roles in checkpoint activation. Three of these proteins-Rad9, Hus1, and Rad1-interact in a heterotrimeric complex (dubbed the 9-1-1 complex), which resembles a PCNA-like sliding clamp, whereas Rad17 is part of a clamp-loading complex that is related to the PCNA clamp loader, replication factor-C (RFC). In response to genotoxic damage, the 9-1-1 complex is loaded around DNA by the Rad17-containing clamp loader. The DNA-bound 9-1-1 complex then facilitates ATR-mediated phosphorylation and activation of Chk1, a protein kinase that regulates S-phase progression, G2/M arrest, and replication fork stabilization. In addition to its role in checkpoint activation, accumulating evidence suggests that the 9-1-1 complex also participates in DNA repair. Taken together, these findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair. 相似文献
19.
Laura A. Lindsey-Boltz Joyce T. Reardon Marc S. Wold Aziz Sancar 《The Journal of biological chemistry》2012,287(43):36123-36131
Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair. 相似文献
20.
The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling. 相似文献