Responding to chromosomal breakage during M-phase: insights from a cell-free system

DNA double strand breaks (DSBs) activate ATM and ATR dependent checkpoints that prevent the onset of mitosis. However, how cells react to DSBs occurring when they are already in mitosis is poorly understood. The Xenopus egg extract has been utilized to study cell cycle progression and DNA damage checkpoints. Recently this system has been successfully used to uncover an ATM and ATR dependent checkpoint affecting centrosome driven spindle assembly. These studies have led to the identification of XCEP63 as major target of this pathway. XCEP63 is a coiled-coil rich protein localized at centrosome essential for proper spindle assembly. ATM and ATR directly phosphorylate XCEP63 on serine 560 inducing its delocalization from centrosome, which in turn delays spindle assembly. This pathway might contribute to regulate DNA repair or mitotic cell survival in the presence of chromosome breakage.     

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     

Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses

In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. In this study, we describe the isolation of a cDNA encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, we have identified the essential replication protein Mcm2 as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 underwent phosphorylation at Ser(92) in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involved both ATM and ATR, but the relative contribution of each kinase depended upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylated Mcm2 directly at Ser(92) in cell-free kinase assays. Immunodepletion of both ATM and ATR abrogated the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts.     

DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint

BACKGROUND: DNA damage during mitosis triggers an ATM kinase-mediated cell cycle checkpoint pathway in yeast and fly embryos that delays progression through division. Recent data suggest that this is also true for mammals. Here we used laser microsurgery and inhibitors of topoisomerase IIalpha to break DNA in various mammalian cells after they became committed to mitosis. We then followed the fate of these cells and emphasized the timing of mitotic progression, spindle structure, and chromosome behavior. RESULTS: We find that DNA breaks generated during late prophase do not impede entry into prometaphase. If the damage is minor, cells complete mitosis on time. However, more significant damage substantially delays exit from mitosis in many cell types. In human (HeLa, CFPAC-1, and hTERT-RPE) cells, this delay occurs during metaphase, after the formation of a bipolar spindle and the destruction of cyclin A, and it is not dependent on a functional p53 pathway. Pretreating cells with ATM kinase inhibitors does not abrogate the metaphase delay due to chromosome damage. Immunofluorescence studies reveal that cells blocked in metaphase by chromosome damage contain one or more Mad2-positive kinetochores, and the block is rapidly overridden when the cells are microinjected with a dominant-negative construct of Mad2 (Mad2deltaC). CONCLUSIONS: We conclude that the delay in mitosis induced by DNA damage is not due to an ATM-mediated DNA damage checkpoint pathway. Rather, the damage leads to defects in kinetochore attachment and function that, in turn, maintain the intrinsic Mad-2-based spindle assembly checkpoint.     

DNA damage activates the SAC in an ATM/ATR-dependent manner, independently of the kinetochore

The DNA damage checkpoint and the spindle assembly checkpoint (SAC) are two important regulatory mechanisms that respond to different lesions. The DNA damage checkpoint detects DNA damage, initiates protein kinase cascades, and inhibits the cell cycle. The SAC relies on kinetochore-dependent assembly of protein complexes to inhibit mitosis when chromosomes are detached from the spindle. The two checkpoints are thought to function independently. Here we show that yeast cells lacking the DNA damage checkpoint arrest prior to anaphase in response to low doses of the DNA damaging agent methyl methane sulfonate (MMS). The arrest requires the SAC proteins Mad1, Mad2, Mad3, Bub1, and Bub3 and works through Cdc20 and Pds1 but unlike the normal SAC, does not require a functional kinetochore. Mec1 (ATR) and Tel1 (ATM) are also required, independently of Chk1 and Rad53, suggesting that Mec1 and Tel1 inhibit anaphase in response to DNA damage by utilizing SAC proteins. Our results demonstrate cross-talk between the two checkpoints and suggest that assembling inhibitory complexes of SAC proteins at unattached kinetochores is not obligatory for their inhibitory activity. Furthermore, our results suggest that there are novel, important targets of ATM and ATR for cell cycle regulation.     

p53 localization at centrosomes during mitosis and postmitotic checkpoint are ATM-dependent and require serine 15 phosphorylation

We recently demonstrated that the p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. Here, we show that ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia-derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from centrosomes by treatments with spindle inhibitors, p53 remained phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, did not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint     

Structure meets function--centrosomes, genome maintenance and the DNA damage response

Centrosomes are cytoplasmic organelles playing a fundamental role in organizing both the interphase cytoskeleton and the bipolar mitotic spindle. In addition, the centrosome has recently come into focus as part of the network that integrates cell cycle arrest and repair signals in response to genotoxic stress--the DNA damage response. One important mediator of this response, the checkpoint kinase Chk1, has been shown to negatively regulate the G(2)/M transition via its centrosomal localization. Moreover, there is growing evidence that a centrosome inactivation checkpoint exists, which utilizes DNA damage-induced centrosome fragmentation or amplification to provoke a "mitotic catastrophe" and eliminate damaged cells. Candidate regulators of this centrosomal checkpoint include the checkpoint kinase Chk2 and its upstream regulators ATM and ATR. In addition, a growing number of other proteins have been implicated in centrosomal regulation of the DNA damage response, e.g. the tumor suppressor p53, the breast cancer susceptibility gene product BRCA1 and mitotic regulators such as Aurora A, Nek2 and the Polo-like kinases Plk1 and Plk3. However, many missing links and discrepancies between different model systems remain.     

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.     

Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint

BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.     

ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication

Mutation of DNA damage checkpoint signaling kinases ataxia telangiectasia-mutated (ATM) or ATM- and Rad3-related (ATR) results in genomic instability disorders. However, it is not well understood how the instability observed in these syndromes relates to DNA replication/repair defects and failed checkpoint control of cell cycling. As a simple model to address this question, we have studied SV40 chromatin replication in infected cells in the presence of inhibitors of ATM and ATR activities. Two-dimensional gel electrophoresis and southern blotting of SV40 chromatin replication products reveal that ATM activity prevents accumulation of unidirectional replication products, implying that ATM promotes repair of replication-associated double strand breaks. ATR activity alleviates breakage of a functional fork as it converges with a stalled fork. The results suggest that during SV40 chromatin replication, endogenous replication stress activates ATM and ATR signaling, orchestrating the assembly of genome maintenance machinery on viral replication intermediates.     

DNA Damage-Induced Accumulation of Centrosomal Chk1 Contributes to its Checkpoint Function

The checkpoint kinase Chk1 is an established transducer of ATR- and ATM-dependent signalling in response to DNA damage. In addition to its nuclear localization, Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B-Cdk1 during unperturbed cell cycles. Here, we demonstrate that DNA damage caused by ultraviolet irradiation or hydroxyurea treatment leads to centrosomal accumulation of endogenous Chk1 in normal human BJ fibroblasts and in ATR- or ATM-deficient fibroblasts. Chemical inhibition of ATR/ATM by caffeine led to enhanced centrosomal Chk1 deposition associated with nuclear Chk1 depletion. In contrast to normal or ATM-deficient fibroblasts, genetically ATR-deficient Seckel-fibroblasts showed detectable constitutive centrosomal accumulation of Chk1 even in the absence of exogenous insults. After DNA damage, the centrosomal fraction of Chk1 was found to be phosphorylated at ATR/ATM phosphorylation sites. Forced immobilization of kinase-inactive but not wild-type Chk1 to centrosomes resulted in a G2/M checkpoint defect. Finally, both DNA damage, and forced centrosomal expression of Chk1 in the absence of genotoxic treatments, induced centrosome amplification in a subset of cells, a phenomenon which could be suppressed by inhibition of ATM/ATR-mediated signaling. Taken together, our results suggest that accumulation of phosphorylated Chk1 at centrosomes constitutes an additional element in the DNA damage response. Centrosomal Chk1 induces G2/M cell cycle arrest and may evoke centrosome amplification, the latter possibly providing a backup mechanism for elimination of cells with impaired DNA damage checkpoints operating earlier during the cell cycle.     

CEP192 interacts physically and functionally with the K63-deubiquitinase CYLD to promote mitotic spindle assembly

CEP192 is a centrosome protein that plays a critical role in centrosome biogenesis and function in mammals, Drosophila and C. elegans.^1-6 Moreover, CEP192-depleted cells arrest in mitosis with disorganized microtubules, suggesting that CEP192’s function in spindle assembly goes beyond its role in centrosome activity and pointing to a potentially more direct role in the regulation of the mitotic microtubule landscape.⁷ To better understand CEP192 function in mitosis, we used mass spectrometry to identify CEP192-interacting proteins. We previously reported that CEP192 interacts with NEDD1, a protein that associates with the γ-tubulin ring complex (γ-TuRC) and regulates its phosphorylation status during mitosis.⁸ Additionally, within the array of proteins that interact with CEP192, we identified the microtubule binding K63-deubiquitinase CYLD. Further analyses show that co-depletion of CYLD alleviates the bipolar spindle assembly defects observed in CEP192-depleted cells. This functional relationship exposes an intriguing role for CYLD in spindle formation and raises the tantalizing possibility that CEP192 promotes robust mitotic spindle assembly by regulating K63-polyubiquitin-mediated signaling through CYLD.     

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.     

Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-independent DNA damage checkpoint

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts.     

Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis.     

Differential regulation of centrosome integrity by DNA damage response proteins

MDC1 and BRIT1 have been shown to function as key regulators in response to DNA damage. However, their roles in centrosomal regulation haven’t been elucidated. In this study, we demonstrated the novel functions of these two molecules in regulating centrosome duplication and mitosis. We found that MDC1 and BRIT1 were integral components of the centrosome that colocalize with γ-tubulin. Depletion of either protein led to centrosome amplification. However, the mechanisms that allow them to maintain centrosome integrity are different. MDC1-depleted cells exhibited centrosome overduplication, leading to multipolar mitosis, chromosome missegregation, and aneuploidy, whereas BRIT1 depletion led to misaligned spindles and/or lagging chromosomes with defective spindle checkpoint activation that resulted in defective cytokinesis and polyploidy. We further illustrated that both MDC1 and BRIT1 were negative regulators of Aurora A and Plk1, two centrosomal kinases involved in centrosome maturation and spindle assembly. Moreover, the levels of MDC1 and BRIT1 inversely correlated with centrosome amplification, defective mitosis, and cancer metastasis in human breast cancer. Together, MDC1 and BRIT1 may function as tumor-suppressor genes, at least in part by orchestrating proper centrosome duplication and mitotic spindle assembly.     

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase and involves ATM

Centrosomes are the principal microtubule organising centres in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, we examined cells that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrested in G2 phase and formed supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurred without an additional DNA replication round and was not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduced DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification was potentiated by Rad54 disruption. Gene targeting of ATM reduced, but did not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. Our data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints.     

DNA Damage to a Single Chromosome End Delays Anaphase Onset

Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as “TIPs”) during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends.     

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.     

The role of Polo-like kinase 1 in the inhibition of centrosome separation after ionizing radiation

Activation of the G2/M cell cycle checkpoint by DNA damage prevents cells from entering mitosis. Centrosome separation is initiated in G2 phase and completed in M phase. This critical process for cell division is targeted by G2/M checkpoint. Here we show that Plk1 signaling plays an important role in regulation of centrosome separation after DNA damage. Constitutively active Plk1 overrides the inhibition of centrosome separation induced by DNA damage. This inhibition is dependent on ATM, but not on Chk2 or Chk1. Nek2 is a key regulator of centrosome separation and is a target of Plk1 in blocking centrosome separation. We found that Plk1 can phosphorylate Nek2 in vitro and interacts with Nek2 in vivo. Down-regulation of Plk1 with RNA interference prevents Nek2-induced centrosome splitting. DNA damage is known to inhibit Plk1 activity. We propose that the DNA damage-induced inhibition of Plk1 leads to inhibition of Nek2 activity and thus prevents centrosome separation.