Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin–TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin–TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin–TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.     

Evidence That the ATR/Chk1 Pathway Maintains Normal Replication Fork Progression during Unperturbed S Phase

If cells are treated with DNA damaging agents or inhibitors that interfere with ongoing DNA replication, the intra-S and S/M checkpoints delay progression through S phase and mitotic entry, respectively, to allow time for DNA repair and replication restart. In vertebrates, these checkpoint responses to replication blocks are largely mediated by the sensor kinase ATR and its major downstream effector kinase Chk1. Increasing evidence suggests that the ATR pathway is also vital in the absence of exogenous stresses, i.e. during “unperturbed” replication. Both ATR and Chk1 are essential proteins in vertebrates, and lack of components of the ATR/Chk1 pathway can result in impaired replication and spontaneous DNA damage. Here we give an overview of how the ATR/Chk1 pathway responds to exogenously blocked replication and then describe evidence for roles of this pathway during replication in an unperturbed S phase.     

Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures

We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2-/- cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1-/- cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.     

Replication stress activates DNA polymerase alpha-associated Chk1

Chk1 contributes to both intra-S and DNA damage checkpoint responses. Here, we show that depletion of DNA Polα and not Polε or Polδ by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Polα and Chk1 causes an accumulation of γ-H2A.X, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Co-depletion of DNA Polα with ATR yields similar phenotypes, suggesting that ATR and Chk1 are epistatic and required for maintenance of genomic integrity following replication stress. Significantly, Chk1 and DNA Polα can be co-immunoprecipated from native cell extracts. Moreover, following replication stress, Polα-associated Chk1 becomes rapidly phosphorylated on Ser345 in a TopBP1 and ATR-dependent manner. Hence, the ability to efficiently phosphorylate Chk1 in the context of DNA Polα complexes is correlated with suppression of DNA damage following replication stress. These findings identify DNA Polα as an important component of the signal transduction cascade that activates the intra-S checkpoint.     

Chk1 regulates the density of active replication origins during the vertebrate S phase

The checkpoint kinase 1 (Chk1) preserves genome integrity when replication is performed on damaged templates. Recently, Chk1 has also been implicated in regulating different aspects of unperturbed S phase. Using mammalian and avian cells with compromised Chk1 activity, we show that an increase in active replicons compensates for inefficient DNA polymerisation. In the absence of damage, loss of Chk1 activity correlates with the frequent stalling and, possibly, collapse of active forks and activation of adjacent, previously suppressed, origins. In human cells, super-activation of replication origins is restricted to pre-existing replication factories. In avian cells, in contrast, Chk1 deletion also correlates with the super-activation of replication factories and loss of temporal continuity in the replication programme. The same phenotype is induced in wild-type avian cells when Chk1 or ATM/ATR is inhibited. These observations show that Chk1 regulates replication origin activation and contributes to S-phase progression in somatic vertebrate cells.     

ATRMec1 phosphorylation-independent activation of Chk1 in vivo

The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks. The current model is that after DNA damage or replication blocks, ATR(Mec1) phosphorylates Chk1 on the non-catalytic C-terminal domain. However, the mechanism of activation of Chk1 and the function of the Chk1 C terminus in vivo remains largely unknown. In this study we used an in vivo assay to examine the role of the C terminus of Chk1 in the response to DNA damage and replication blocks. The conserved ATR(Mec1) phosphorylation sites were essential for the checkpoint response to DNA damage and replication blocks in vivo; that is, that mutation of the sites caused lethality when DNA replication was stalled by hydroxyurea. Despite this, loss of the ATR(Mec1) phosphorylation sites did not change the kinase activity of Chk1 in vitro. Furthermore, a single amino acid substitution at an invariant leucine in a conserved domain of the non-catalytic C terminus restored viability to cells expressing the ATR(Mec1) phosphorylation site-mutated protein and relieved the requirement of an upstream mediator for Chk1 activation. Our findings show that a single amino acid substitution in the C terminus, which could lead to an allosteric change in Chk1, allows it to bypass the requirement of the conserved ATR(Mec1) phosphorylation sites for checkpoint function.     

Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

In yeasts, the replication protein Cdc6/Cdc18 is required for the initiation of DNA replication and also for coupling S phase with the following mitosis. In metazoans a role for Cdc6 has only been shown in S phase entry. Here we provide evidence that human Cdc6 (HuCdc6) also regulates the onset of mitosis, as overexpression of HuCdc6 in G(2) phase cells prevents entry into mitosis. This block is abolished when HuCdc6 is expressed together with a constitutively active Cyclin B/CDK1 complex or with Cdc25B or Cdc25C. An inhibitor of Chk1 kinase activity, UCN-01, overcomes the HuCdc6 mediated G(2) arrest indicating that HuCdc6 blocks cells in G(2) phase via a checkpoint pathway involving Chk1. When HuCdc6 is overexpressed in G(2), we detected phosphorylation of Chk1. Thus, HuCdc6 can trigger a checkpoint response, which could ensure that all DNA is replicated before mitotic entry. We also present evidence that the ability of HuCdc6 to block mitosis may be regulated by its phosphorylation.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

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.     

Activation of the DNA damage checkpoint in mutants defective in DNA replication initiation

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.     

Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.     

Unleashing Chk1 in cancer therapy

The checkpoint kinase 1 (Chk1) is one of the major players in the signal transduction pathway set in motion in response to DNA damage which activates different cell cycle checkpoints including the G₁/S, the intra-S, G₂-M and the mitotic spindle checkpoint, contributing to the maintenance of genomic stability. Chk1 is considered a good molecular target to inhibit, in combination with other anticancer agents, to increase the sensitivity of treatment, especially in tumors with a defective G₁ checkpoint. Experimental evidence highlights the essential role of Chk1 in normal and cancer cells even under unstressed conditions, especially in controlling DNA replication and cell division. This review looks at the main functions of Chk1 and the data on Chk1 inhibitors at their preclinical and clinical development are reported. This information may suggest novel approaches for new treatments with Chk1 inhibitors in combination with anticancer agents or as single agents. The emergent synthetic lethality approach may help define the genetic background features where Chk1 inhibitors alone could be very effective.     

Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1

The G2 DNA damage checkpoint delays mitotic entry via the upregulation of Wee1 kinase and the downregulation of Cdc25 phosphatase by Chk1 kinase, and resultant inhibitory phosphorylation of Cdc2. While checkpoint activation is well understood, little is known about how the checkpoint is switched off to allow cell cycle re-entry. To identify proteins required for checkpoint release, we screened for genes in Schizosaccharomyces pombe that, when overexpressed, result in precocious mitotic entry in the presence of DNA damage. We show that overexpression of the type I protein phosphatase Dis2 sensitises S. pombe cells to DNA damage, causing aberrant mitoses. Dis2 abrogates Chk1 phosphorylation and activation in vivo, and dephosphorylates Chk1 and a phospho-S345 Chk1 peptide in vitro. dis2Delta cells have a prolonged chk1-dependent arrest and a compromised ability to downregulate Chk1 activity for checkpoint release. These effects are specific for the DNA damage checkpoint, because Dis2 has no effect on the chk1-independent response to stalled replication forks. We propose that inactivation of Chk1 by Dis2 allows mitotic entry following repair of DNA damage in the G2-phase.     

BRCA1-dependent Chk1 phosphorylation triggers partial chromatin disassociation of phosphorylated Chk1 and facilitates S-phase cell cycle arrest

Chk1 phosphorylation by the PI3-like kinases ATR and ATM is critical for its activation and its role in prevention of premature mitotic entry in response to DNA damage or stalled replication. The breast and ovarian tumor suppressor, BRCA1, is among several checkpoint mediators that are required for Chk1 activation by ATM and ATR. Previously we showed that BRCA1 is necessary for Chk1 phosphorylation and activation following ionizing radiation. BRCA1 has been implicated in S-phase checkpoint control yet its mechanism of action is not well characterized. Here we report that BRCA1 is critical for Chk1 phosphorylation in response to inhibition of replication by either cisplatin or hydroxyurea. While Chk1 phosphorylation of S317 is fully dependent on BRCA1, additional proteins may mediate S345 phosphorylation at later time points. In addition, we show that a subset of phosphorylated Chk1 is released from the chromatin in a BRCA1-dependent manner which may lead to the phosphorylation of Chk1 substrate, Cdc25C, on S216 and to S-phase checkpoint activation. Inhibition of Chk1 kinase by UCN-01 or expression of Chk1 phosphorylation mutants in which the serine residues were substituted with alanine residues abrogates BRCA1-dependent cell cycle arrest in response replication inhibition. These data reveal that BRCA1 facilitates Chk1 phosphorylation and its partial chromatin dissociation following replication inhibition that is likely to be required for S-phase checkpoint signaling.     

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.     

Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC signifi-cantly delayed the progression of the S phase and caused the phosphorylation of the Chk 1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern II was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-S-phase checkpoint blocks the DNA replication progression in the late stage of S phase.     

Hsp90 inhibition depletes Chk1 and sensitizes tumor cells to replication stress

DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled replication forks, and participates in G2 arrest. In this study, we show that Chk1 interacts with Hsp90, a molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins known as clients. Consistent with Chk1 being an Hsp90 client, we also found that Chk1 but not Chk2 is destabilized in cells treated with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated Chk1 loss blocked the ability of Chk1 to target Cdc25A for proteolytic destruction, demonstrating that the Chk1 signaling pathway was disrupted in the 17-AAG-treated cells. Finally, 17-AAG-mediated disruption of Chk1 activation dramatically sensitized various tumor cells to gemcitabine, an S phase-active chemotherapeutic agent. Collectively, our studies identify Chk1 as a novel Hsp90 client and suggest that pharmacologic inhibition of Hsp90 may sensitize tumor cells to chemotherapeutic agents by disrupting Chk1 function during replication stress.     

Human claspin is required for replication checkpoint control

Claspin is a newly identified protein that regulates Chk1 activation in Xenopus. In the present study we investigated the role of human Claspin in the DNA damage/replication checkpoint in mammalian cells. We observed that human Claspin is a cell cycle regulated protein that peaks at S/G2 phase. Claspin localizes in the nuclei, but it only associates with Chk1 following replication stress or other types of DNA damage. In addition, Claspin is phosphorylated in response to replication stress, and this phosphorylation appears to be required for its association with Chk1. Moreover, Claspin interacts with the checkpoint proteins ATR and Rad9. Given that both the ATR and Rad9-Rad1-Hus1 complexes are involved in Chk1 activation, it is possible that Claspin works as an adaptor molecule bringing these molecules together. Using small interfering RNA technology, we have shown that down-regulation of Claspin expression inhibits Chk1 activation in response to replication stress. More importantly, down-regulation of Claspin augments the premature chromatin condensation induced by hydroxyurea, inhibits the UV-induced reduction of DNA synthesis, and decreases cell survival. Taken together, these data imply a potentially critical role for Claspin in replication checkpoint control in mammalian cells.