Regulation of intra-S phase checkpoint by ionizing radiation (IR)-dependent and IR-independent phosphorylation of SMC3

Structure maintenance of chromosome 1 (SMC1) is phosphorylated by ataxia telangiectasia-mutated (ATM) in response to ionizing radiation (IR) to activate intra-S phase checkpoint. A role of CK2 in DNA damage response has been implicated in many previous works, but the molecular mechanism for its activation is not clear. In the present work, we report that SMC3 is phosphorylated at Ser-1067 and Ser-1083 in vivo. Ser-1083 phosphorylation is IR-inducible, depends on ATM and Nijmegen breakage syndrome 1 (NBS1), and is required for intra-S phase checkpoint. Interestingly, Ser-1067 phosphorylation is constitutive and is not induced by IR but also affects intra-S phase checkpoint. Phosphorylation of Ser-1083 is weakened in cells expressing S1067A mutant, suggesting interplay between Ser-1067 and Ser-1083 phosphorylation in DNA damage response. Consistently, small interfering RNA knockdown of CK2 leads to attenuated phosphorylation of Ser-1067 as well as intra-S phase checkpoint defect. Our data provide evidence that phosphorylation of a core cohesin subunit SMC3 by ATM plays an important role in DNA damage response and suggest that a constitutive phosphorylation by CK2 may affect intra-S phase checkpoint by modulating SMC3 phosphorylation by ATM.     

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G?/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G?/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G?/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G?/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.     

A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints

ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.     

Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G₂/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G₂/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G₂/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G₂/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.     

ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling

The phosphatidyl inositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) regulate parallel damage response signalling pathways. ATM is reported to be activated by DNA double-strand breaks (DSBs), whereas ATR is recruited to single-stranded regions of DNA. Although the two pathways were considered to function independently, recent studies have demonstrated that ATM functions upstream of ATR following exposure to ionising radiation (IR) in S/G2. Here, we show that ATM phosphorylation at Ser1981, a characterised autophosphorylation site, is ATR-dependent and ATM-independent following replication fork stalling or UV treatment. In contrast to IR-induced ATM-S1981 phosphorylation, UV-induced ATM-S1981 phosphorylation does not require the Nbs1 C-terminus or Mre11. ATR-dependent phosphorylation of ATM activates ATM phosphorylation of Chk2, which has an overlapping function with Chk1 in regulating G2/M checkpoint arrest. Our findings provide insight into the interplay between the PIKK damage response pathways.     

Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition

Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.     

A Functional Link Between SIRT1 Deacetylase and NBS1 in DNA Damage Response

In mammalian cells, DNA is often subjected to stresses such as ionizing radiation (IR) and ultraviolet light that can induce DNA double strand breaks (DSBs). In response to DNA DSBs, mammalian cells activate a rapid phosphorylation signaling cascade through the protein kinases, Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-Related (ATR).1 Many well-characterized DNA repair factors are phosphorylated by ATM in response to DSBs, and the sequential phosphorylation of some of these factors, including NBS1, delay cell cycle progression (checkpoint arrest) to allow time for DNA damage repair.2 Results from a new study suggest that phosphorylation of NBS1 is regulated by the acetylation status of the protein, which is modulated by SIRT1 deacetylase.     

The roles of p27Kip1 and DNA damage signalling in the chemotherapy‐induced delayed cell cycle checkpoint

DNA lesions trigger the DNA damage response (DDR) machinery, which protects genomic integrity and sustains cellular survival. Increasing data underline the significance of the integrity of the DDR pathway in chemotherapy response. According to a recent work, persistent exposure of A549 lung carcinoma cells to doxorubicin induces an initial DDR‐dependent checkpoint response, followed by a later DDR‐independent, but p27^Kip1‐dependent one. Prompted by the above report and to better understand the involvement of the DDR signaling after chemotherapeutic stress, we examined the potential role of the canonical DDR pathway in A549 cells treated with doxorubicin. Exposure of A549 cells, prior to doxorubicin treatment, to ATM, ATR and DNA‐PKcs inhibitors either alone or in various combinations, revealed that the earlier documented two‐step response was DDR‐dependent in both steps. Notably, inhibition of both ATM and ATR or selective inhibition of ATM or DNA‐PKcs resulted in cell‐cycle re‐entry despite the increased levels of p27^Kip1 at all time points analyzed. We further investigated the regulation of p27^Kip1 protein levels in the particular setting. Our results showed that the protein status of p27^Kip1 is mainly determined by p38‐MAPK, whereas the role of SKP2 is less significant in the doxoroubicin‐treated A549 cells. Cumulatively, we provide evidence that the DNA damage signaling is responsible for the prolonged cell cycle arrest observed after persistent chemotherapy‐induced genotoxic stress. In conclusion, precise identification of the molecular mechanisms that are activated during the chemotherapeutic cycles could potentially increase the sensitization to the therapy applied.     

The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2‐7 subcomplex of the replicative Cdc45‐MCM‐GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL. Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.     

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.     

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation,fork restart,homologous recombination and mitotic catastrophe

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.     

Elevated cyclin G2 expression intersects with DNA damage checkpoint signaling and is required for a potent G2/M checkpoint arrest response to doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.     

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.     

ATR regulates hexavalent chromium-induced S-phase checkpoint through phosphorylation of SMC1

Hexavalent chromium (Cr[VI]) is an industrial waste product known to cause nasal and lung cancer in exposed workers. Intracellularly, Cr[VI] undergoes a series of enzymatic reductions resulting in the formation of reactive chromate intermediates and oxygen free radicals. These metabolites react with DNA to cause numerous types of genomic lesions, but the cellular response to these genotoxic insults is poorly understood. Recently, we demonstrated that in response to DNA damage induced by Cr[VI], an ataxia-telangiectasia mutated (ATM) and structural maintenance of chromosomal protein 1 (SMC1)-dependent S-phase checkpoint is activated. Interestingly, this checkpoint response was only ATM-dependent in cells exposed to low doses of Cr[VI], we demonstrate that the ATM and Rad3 related kinase, ATR, is required to activate the S-phase checkpoint. In response to all doses of Cr[VI], ATR is activated and phosphorylates SMC1 to facilitate the checkpoint. Further, chromatin binding ability of Rad17 is required for this process. Taken together, these results indicate that the Rad17-ATR-SMC1 pathway is essential for Cr[VI]-induced S-phase checkpoint activation.     

NOD2 agonist murabutide alleviates radiation-induced injury through DNA damage response pathway mediated by ATR

Injury-induced by ionizing radiation (IR) severely reduces the quality of life of victims. The development of radiation protectors is regarded as one of the most resultful strategies to alleviate damages caused by IR exposure. In the present study, we investigated the radioprotective effects of the agonist of nucleotide-binding-oligomerization-domain-containing proteins 2 called murabutide (MBD) and clarified the potential mechanisms. Our results showed that the pretreatment with MBD effectively protected cultured cells and mice against IR-induced toxicity and the pretreatment with MBD in vitro and in vitro also inhibited apoptosis caused by IR exposure. The downregulation of γ-H2AX and the upregulation of ATR signaling pathways by MBD treatment indicated that the radioprotective effects of MBD were due to the stimulation of DNA damage response (DDR) pathway to repair DNA double-strand breaks caused by IR exposure. As the radioprotective effects of MBD were diminished by the ATR selective inhibitor rather than the ATM inhibitor, ATR pathway was confirmed to be a more crucial checkpoint pathway in mediating the stimulation of DDR pathway by MBD. Taken together, our data provide a novel and effective protector to relieve the injury induced by IR exposure.     

Centrosomal localization of DNA damage checkpoint proteins

During mitosis, the phosphatidylinositol-3 (PI-3) family-related DNA damage checkpoint kinases ATM and ATR were found on the centrosomes of human cells. ATRIP, an interaction partner of ATR, as well as Chk1 and Chk2, the downstream targets of ATR or ATM, were also localized to the centrosomes. Surprisingly, the DNA-PK inhibitor vanillin enhanced the level of ATM on centrosomes. Accordingly, DNA-PKcs, the catalytic subunit of DNA-PK, was also found on the centrosomes. Vanillin altered the phosphorylation of Chk2 in the centrosomes and in whole cell extracts. Nucleoplasmic ATM co-immunoprecipitated with Ku70/86, the DNA binding subunits of DNA-PK, while vanillin diminished this association. Vanillin did not affect microtubule polymerization at the centrosomes but, surprisingly, caused a transient enhancement of alpha-tubulin foci in the nucleus. Interestingly, gamma-tubulin was also present in the nucleus and co-immunoprecipitated with ATR or BRCA1. DNA damage led to a reduction of the mentioned checkpoint proteins on the centrosomes but increased the level of gamma-tubulin at this organelle. Taken together, these results indicate that DNA damage checkpoint proteins may control the formation of gamma-tubulin and/or the kinetics of microtubule formation at the centrosomes, and thereby couple them to the DNA damage response.     

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G₁ phase to G₂ and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G₁ cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.