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.     

Asymmetric localization of the CDC25B phosphatase to the mother centrosome during interphase

The Cyclin-Dependent Kinase (CDK)-activating phosphatase CDC25B, localises to the centrosomes where its activity is both positively and negatively regulated by several kinases including Aurora A and CHK1. Our recent data also demonstrate a role for CDC25B in the centrosome duplication cycle and microtubule nucleation in interphase that appears to involve the recruitment of γ-tubulin to the centrosomes. In the present study, we report that CDC25B, along with CHK1, CDK1 and WEE1, localise asymmetrically around the mother centrosome from S to G2-phases, and gradually become evenly distributed to the two centrosomes by late G2 phase, concomitant with centrosome maturation. We further demonstrate that siRNA inhibition of CDC25B results in an accumulation of cells in G2 phase with two separated centrosomes, each containing only a single centriole, suggesting a requirement for CDC25B in centriole duplication. We propose that the localisation of key cell cycle regulators to the mother centrosome ensures synchrony between the centrosome duplication and cell division cycles.     

p21 inhibits Thr161 phosphorylation of Cdc2 to enforce the G2 DNA damage checkpoint

The cyclin-dependent kinase inhibitor p21 is required for a sustained G(2) arrest after activation of the DNA damage checkpoint. Here we have addressed the mechanism by which p21 can contribute to this arrest in G(2). We show that p21 blocks the activating phosphorylation of Cdc2 on Thr(161). p21 does not interfere with the dephosphorylation of two inhibitory phosphorylation sites on Cdc2, Thr(14) and Tyr(15), indicating that p21 targets a different event in Cdc2 activation as the well described DNA damage checkpoint pathway involving Chk1 and Cdc25C. Taken together our data show that a cell is equipped with at least two independent pathways to ensure efficient inhibition of Cdc2 activity in response to DNA damage, influencing both positive and negative regulatory phosphorylation events on Cdc2.     

DNA damage during reoxygenation elicits a Chk2-dependent checkpoint response

Due to the abnormal vasculature of solid tumors, tumor cell oxygenation can change rapidly with the opening and closing of blood vessels, leading to the activation of both hypoxic response pathways and oxidative stress pathways upon reoxygenation. Here, we report that ataxia telangiectasia mutated-dependent phosphorylation and activation of Chk2 occur in the absence of DNA damage during hypoxia and are maintained during reoxygenation in response to DNA damage. Our studies involving oxidative damage show that Chk2 is required for G2 arrest. Following exposure to both hypoxia and reoxygenation, Chk2-/- cells exhibit an attenuated G2 arrest, increased apoptosis, reduced clonogenic survival, and deficient phosphorylation of downstream targets. These studies indicate that the combination of hypoxia and reoxygenation results in a G2 checkpoint response that is dependent on the tumor suppressor Chk2 and that this checkpoint response is essential for tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5′ to 3′ DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5′ to 3′ exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5′ to 3′ helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.Key words: yeast, RAD27, RAD9, RAD53, Okazaki fragment processing, DNA replication, exo1     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.     

The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint

Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and we delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a nonspecific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1.     

Sensing, signaling, and responding to DNA damage: organization of the checkpoint pathways in mammalian cells

The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate cellular responses to genotoxic conditions. Unlike typical signal transduction mechanisms that respond to one or a few stimuli, checkpoints can be activated by a broad spectrum of extrinsically or intrinsically derived DNA damage or replication interference. Recent investigations have shed light on how the damage and replication checkpoints are able to respond to such diverse stimuli. The activation of checkpoints not only attenuates cell cycle progression but also facilitates DNA repair and recovery of faltered replication forks, thereby preventing DNA lesions from being converted to inheritable mutations. Recently, more checkpoint targets from the cell cycle and DNA replication apparatus have been identified, revealing the increasing complexity of the checkpoint control of the cell cycle. In this article, we discuss current models of the DNA damage and replication checkpoints and highlight recent advances in the field.     

Combination of the 6-thioguanine and disulfiram/Cu synergistically inhibits proliferation of triple-negative breast cancer cells by enhancing DNA damage and disrupting DNA damage checkpoint

Because of the lack of specific molecular targeted therapies, triple-negative breast cancer (TNBC) has high tumour recurrence and metastasis rates. It is urgent to develop novel chemotherapeutic strategies to improve patient survival. DNA damaging agents have been shown to sensitize cancer to genotoxic chemotherapies. We first found that 6-thioguanine (6-TG) can activate the NF-кB signalling pathway. Our results showed that NF-кB signalling was reduced when cells were treated with 6-TG/disulfiram (DSF)/Cu. DSF/Cu enhanced the 6-TG-mediated inhibition of proliferation. 6-TG/DSF/Cu inhibited cell cycle progression, causing cell cycle arrest in the S phase and G2/M phase. Moreover, the combined effect of 6-TG and DSF/Cu induced apoptosis, and either agent alone was able to induce apoptosis. The accumulation of γH2A indicated that DSF/Cu increased the DNA damage induced by 6-TG. Combined treatment with 6-TG and DSF/Cu synergistically reduced the levels of both phosphorylated and total ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR), suggesting that DSF/Cu promoted 6-TG-induced DNA damage by suppressing ATR protein kinases, therefore enhancing cell apoptosis. In conclusion, we demonstrate that the combination of 6-TG and DSF/Cu exerted a significant synergistic antitumour effect on human TNBC in vitro and in vivo by enhancing DNA damage and disrupting DNA damage checkpoints. We propose that this combination therapy could be a novel strategy for the treatment of TNBC.     

DNA replication defects, spontaneous DNA damage, and ATM-dependent checkpoint activation in replication protein A-deficient cells

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways.     

Cohesin defects lead to premature sister chromatid separation,kinetochore dysfunction,and spindle-assembly checkpoint activation

Scc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules.     

Oxidative stress-induced DNA damage of mouse zygotes triggers G2/M checkpoint and phosphorylates Cdc25 and Cdc2

In vitro fertilized (IVF) embryos show both cell cycle and developmental arrest. We previously showed oxidative damage activates the ATM?→?Chk1?→?Cdc25B/Cdc25C cascade to mediate G2/M cell cycle arrest for repair of hydrogen peroxide (H₂O₂)-induced oxidative damage in sperm. However, the mechanisms underlying the developmental delay of zygotes are unknown. To develop a model of oxidative-damaged zygotes, we treated mouse zygotes with different concentrations of H₂O₂ (0, 0.01, 0.02, 0.03, 0.04, 0.05 mM), and evaluated in vitro zygote development, BrdU incorporation to detect the duration of S phase. We also examined reactive oxygen species level and used immunofluorescence to detect activation of γH2AX, Cdc2, and Cdc25. Oxidatively damaged zygotes showed a delay in G2/M phase and produced a higher level of ROS. At the same time, γH2AX was detected in oxidatively damaged zygotes as well as phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15). Our study indicates that oxidative stress-induced DNA damage of mouse zygotes triggers the cell cycle checkpoint, which results in G2/M cell cycle arrest, and that phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15) participate in activating the G2/M checkpoint.     

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.     

Human CDC25B and CDC25C differ by their ability to restore a functional checkpoint response after gene replacement in fission yeast

In fission yeast, inactivation of the Cdc25 phosphatase by checkpoint kinases participates in the signaling cascade that temporarily stops cell cycle progression after DNA damage. In human, CDC25B and C are also known to be targeted by a similar checkpoint machinery. We have examined by homologous recombination, whether CDC25B and CDC25C were able to substitute for the function of fission yeast Cdc25. We demonstrate that (i) CDC25B and C efficiently replace Cdc25 for vegetative growth, (ii) CDC25C is able to restore a functional checkpoint in response to ionizing radiation in both a Chk1- and Cds1-dependent manner, (iii) CDC25B and C are equally efficient in the response to UV irradiation, CDC25B being only dependent on Chk1, while CDC25C depends on both Chk1 and Cds1, and (iv) CDC25C is able to restore a functional DNA replication checkpoint induced by hydroxyurea in a Cds1-dependent manner. The consequences of these findings on our current view of the checkpoint cascade are discussed.     

Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA

The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17–Rfc2–Rfc3–Rfc4–Rfc5) complex and an RHR heterotrimer (Rad1–Hus1–Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5′ recessed ends whereas RFC preferred 3′ recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5′ recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.     

Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint

Dovitinib (TKI258; formerly CHIR‐258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G₂/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single‐cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G₂ arrest similar to the G₂ DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ‐H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G₂ arrest could be overcome by abrogation of the G₂ DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib‐mediated G₂ cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP‐ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.     

A neuroprotective role for the DNA damage checkpoint in tauopathy

ATM and p53, effectors of the DNA damage checkpoint, are generally considered pro-apoptotic in neurons. We show that DNA damage and checkpoint activation occurs in postmitotic neurons in animal models of tauopathy, neurodegenerative disorders that include Alzheimer's disease. Surprisingly, checkpoint attenuation potently increases neurodegeneration through aberrant cell cycle re-entry of postmitotic neurons. These data suggest an unexpected neuroprotective role for the DNA damage checkpoint in tauopathies.     

A new layer of regulation is required to silence the DNA damage checkpoint

Comment on: Wood MD, et al. Cell Cycle 2010; 9:3354-64.     

Proteins in the nutrient-sensing and DNA damage checkpoint pathways cooperate to restrain mitotic progression following DNA damage

Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA.