Depletion Of ATR Research Articles

ATM and ATR Signaling Regulate the Recruitment of Human Telomerase to Telomeres

The yeast homologs of the ATM and ATR DNA damage response kinases play key roles in telomerase-mediated telomere maintenance, but the role of ATM/ATR in the mammalian telomerase pathway has been less clear. Here, we demonstrate the requirement for ATM and ATR in the localization of telomerase to telomeres and telomere elongation in immortal human cells. Stalled replication forks increased telomerase recruitment in an ATR-dependent manner. Furthermore, increased telomerase recruitment was observed upon phosphorylation of the shelterin component TRF1 at an ATM/ATR target site (S367). This phosphorylation leads to loss of TRF1 from telomeres and may therefore increase replication fork stalling. ATM and ATR depletion reduced assembly of the telomerase complex, and ATM was required for telomere elongation in cells expressing POT1ΔOB, an allele of POT1 that disrupts telomere-length homeostasis. These data establish that human telomerase recruitment and telomere elongation are modulated by DNA-damage-transducing kinases.

ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.

Abstract 794: The ATR inhibitor VE-821 in combination with the novel topoisomerase I inhibitor LMP-400 selectively kills cancer cells by disabling DNA replication initiation and fork elongation

Abstract Camptothecin, a specific topoisomerase I inhibitor is a potent anticancer drug, especially against solid tumors. This agent produces well-characterized double-strand breaks upon collision of replication forks with topoisomerase I cleavage complexes. In an attempt to improve its efficacy, we conducted a synthetic lethal siRNA screening using a library that targets nearly 7000 human genes. Depletion of ATR, the main transducer of replication stress-induced DNA damage response exacerbated cytotoxic response to both camptothecin and the indenoisoquinoline LMP-400, a novel class of topoisomerase inhibitors in clinical trial. Inhibition of ATR by the recently developed specific inhibitor VE-821 induced synergistic antiproliferative activity when combined with either topoisomerase inhibitor. Cytotoxicity induced by the combination with LMP-400 was greater than with camptothecin. Using single cell analysis and DNA fiber spread, we show that VE-821 abrogated the S-phase checkpoint, restored origin firing and replication fork progression. Moreover, the combination of a topoisomerase inhibitor with VE-821 inhibited the phosphorylation of ATR and ATR-mediated Chk1 phosphorylation but strongly induced γH2AX. Single cell analysis revealed that γH2AX pattern changed overtime from well-defined focus to a pan-nuclear staining. The change in γH2AX pattern can be useful as a predictive biomarker to evaluate the efficacy of therapy. The key implication of our work is the clinical rationale it provides to evaluate the combination of indenoisoquinoline topoisomerase I inhibitors with ATR inhibitors. Citation Format: Rozenn Jossé, Scott E. Martin, Rajarshi Guha, Pinar Ormanoglu, Thomas Pfister, Joel Morris, James H. Doroshow, Yves Pommier. The ATR inhibitor VE-821 in combination with the novel topoisomerase I inhibitor LMP-400 selectively kills cancer cells by disabling DNA replication initiation and fork elongation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 794. doi:10.1158/1538-7445.AM2014-794

The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice.

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway.

ATR suppresses apoptosis after UVB irradiation by controlling both translesion synthesis and alternative tolerance pathways.

Ultraviolet (UV) light can stall replication forks owing to the formation of bulky lesions in the DNA. Replication across these blocking lesions occurs through translesion DNA synthesis, and cells activate the ATR damage responses to UV. However, it remains unclear whether lesion bypass requires the replication checkpoint because ATR is not necessary for PCNA ubiquitylation. We observed that ATR knockdown by siRNA increased replication stress and promoted early induction of apoptosis following UVB irradiation in SV40-immortalized human cells, including cells from XP-V and XP-C patients. XP-V cells were further sensitized by the silencing, indicating that DNA polymerase η (Pol η) remains active despite ATR control. However, following UVB irradiation, ATR-depleted cells were unable to achieve mitosis, as would be expected after the loss of a DNA checkpoint control. Thus, ATR also regulates replication arrest recovery following UVB-induced damage, independently of Pol η, in SV40-immortalized cell lines. The ATR-mediated DNA damage response regulates replication and different tolerance pathways, and in these cells, ATR depletion induces replication catastrophe, which contributes to explain the potential of ATR inhibition to protect against UVB-induced carcinogenesis.

Abstract 1011: Induction of ‘BRCAness’ by treatment with histone deacetylase inhibitors sensitizes human triple negative breast cancer cells to PARP inhibitor and cisplatinum.

Abstract During DNA damage response (DDR), stability and activity of ATR and CHK1 is essential for the cell cycle arrest and subsequent DNA repair through BRCA1-regulated homologous recombination (HR). We have previously reported that ATR and CHK1 are hsp90 client proteins and treatment with hsp90 inhibitor sensitizes cancer cells to DNA damage. In the present studies, we determined that treatment with pan-HDAC inhibitors vorinostat (VS) and panobinostat (PS) induced hyper-acetylation and inhibition of chaperone function of nuclear hsp90, leading to proteasomal degradation and depletion of ATR, CHK1 and BRCA1. This led to inhibition of DDR and DNA repair following ionizing radiation (IR) through destabilization of ATR-CHK1 and BRCA1 proteins. Specific siRNA-mediated knockdown of HDAC3 but not of HDAC1 or HDAC2 also induced hyper-acetylation of the nuclear hsp90 and depletion of ATR and CHK1, indicating that among the class I HDACs, HDAC3 is the deacetylase for the nuclear hsp90. We next determined whether, by depleting DDR proteins and BRCA1 and inducing ‘BRCAness’, treatment with VS would sensitize the triple negative breast cancer (TNBC) SUM159PT and MB-231 (lacking BRCA1 mutation) as well as BRCA1 mutant HCC1937 cells to the PARP inhibitor ABT888 (veliparib). Indeed, combined treatment with VS or PS and ABT888 (10 to 20 μM) for 48 hours synergistically induced apoptosis (combination indices (CI) by isobologram analysis being < 1.0) of TNBC cells with (HCC1937) or without BRCA1 mutation (MB-231 and SUM159PT cells). As compared to treatment with each agent alone, co-treatment with VS and ABT888 also induced significantly more DNA strand breaks, as demonstrated by higher γ-H2AX levels. Combined treatment also induced markedly greater tail moment, as determined by the comet assay that evaluates the SYBR® Green-stained DNA tails by fluorescent microscopy. Co-treatment with VS and ABT888 also induced more BH3 domain-only pro-death protein BIM, while knockdown of BIM by shRNA significantly reduced the apoptosis induced by co-treatment with VS and ABT888. It is noteworthy that treatment with VS also sensitized the TNBC cells to cisplatin (2.0 to 10 μM)-induced apoptosis. Moreover, co-treatment with VS and ciplatin synergistically induced apoptosis of TNBC cells (CIs < 1.0). These findings indicate that treatment with pan-HDAC inhibitors VS or PS creates ‘BRCAness’, and in combination with a PARP inhibitor or cisplatin synergistically induces apoptosis in human TNBC cells. These findings support a compelling rationale to confirm whether the combination of VS or PS with ABT888 and cisplatin would be a highly active treatment of human TNBC, irrespective of its expression of mutant BRCA1. Citation Format: Warren C. Fiskus, Rekha Rao, Soumyasri Das Gupta, Lata Chauhan, Kapil N. Bhalla. Induction of ‘BRCAness’ by treatment with histone deacetylase inhibitors sensitizes human triple negative breast cancer cells to PARP inhibitor and cisplatinum. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1011. doi:10.1158/1538-7445.AM2013-1011

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which is mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that are separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.

Abstract S3-7: Treatment with Histone Deacetylase Inhibitors Creates ‘BRCAness’ and Sensitizes Human Triple Negative Breast Cancer Cells to PARP Inhibitors and Cisplatin

Abstract DNA damage induces DNA damage response (DDR), which regulates cell cycle transit, DNA repair and apoptosis. During DDR, stability and activity of ATR and CHK1 is essential for the cell cycle arrest and subsequent DNA repair through homologous recombination (HR) in which BRCA1 protein is involved. We have previously reported that ATR, CHK1 and BRCA1 are hsp90 client proteins and treatment with hsp90 inhibitor sensitizes cancer cells to DNA damage. We have also previously shown that treatment with pan-HDAC inhibitors, including vorinostat (VS) and panobinostat (PS), induces hyperacetylation of hsp90, thereby inhibiting its chaperone function. In the present studies we determined that treatment with VS or PS induced hyper-acetylation and inhibition of chaperone function of nuclear hsp90, leading to proteasomal degradation and depletion of ATR, CHK1 and BRCA1. This led to inhibition of DDR and DNA repair following ionizing radiation (IR) through destabilization of ATR-CHK1 and BRCA1 proteins. Based on this, we hypothesized that treatment with VS or PS would create ‘BRCAness’ in breast cancer cells. Specific siRNA-mediated knockdown of HDAC3 but not of HDAC1 or HDAC2 also induced hyper-acetylation of the nuclear hsp90 and depletion of ATR and CHK1, indicating that among the class I HDACs, HDAC3 is the deacetylase for the nuclear hsp90. We next determined whether, by depleting DDR proteins and BRCA1 and inducing ‘BRCAness’, treatment with VS would sensitize the triple negative breast cancer (TNBC) SUM59PT, MB-231 and HCC1937 cells to the PARP inhibitor ABT888 (veliparib). Indeed, combined treatment with VS or PS with ABT888 (10 to 20 μM) for 48 hours synergistically induced apoptosis (Combination indices by isobologram analysis being < 1.0) of TNBC cells with (HCC1937) or without BRCA1 mutation (MB-231 and SUM159PT cells). As compared to treatment with each agent alone, co-treatment with VS and ABT888 also induced significantly more DNA strand breaks, as demonstrated by higher γ-H2AX levels. Combined treatment also induced markedly greater tail moment, as determined by the Comet assay that evaluates the SYBR green-stained DNA tails by fluorescent microscopy. Co-treatment with VS and ABT888 also induced more BH3 domain-only pro-death protein BIM, while knock-down of BIM by shRNA significantly reduced the apoptosis induced by co-treatment with VS and ABT888. It is noteworthy that treatment with VS also sensitized the TNBC cells to cisplatin (2.0 to 10 μM)-induced apoptosis. Moreover, co-treatment with VS and ciplatin synergistically induced apoptosis of TNBC cells (CIs < 1.0). These findings indicate that treatment with pan-HDAC inhibitors VS or PS creates BRCAness, and in combination with a PARP inhibitor or cisplatin synergistically induces apoptosis in human TNBC cells. These findings support a compelling rationale to confirm whether the combination of VS or PS with ABT888 and cisplatin would be a highly active treatment in the in vivo models of human TNBC cells, irrespective of their expression of mutant BRCA1. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S3-7.

Abstract 4698: Inhibition of histone deacetylase (HDAC) 3 induces hyperacetylation and inhibition of nuclear heat shock protein (hsp) 90 leading to depletion of ATR and CHK1 with sensitization to DNA damage in breast and cervical cancer cells

Abstract We have recently reported that in addition to checkpoint kinase 1 (CHK1), ataxia-telangiectasia mutated and Rad3- related (ATR) is chaperoned by hsp90. Treatment of breast and cervical cancer cells with hsp90 inhibitor induces proteasomal degradation and depletion of ATR and CHK1. This results in impairment of the DNA damage response (DDR) and causes sensitization to α-irradiation and replication stress due to hydroxyurea (Mol Cancer Ther 10:1194, 3011). In the present studies, we determined that treatment with pan-histone deacetylase (HDAC) inhibitor panobinostat (20 to 50 nM) or vorinostat (0.5 to 2 μM) induced polyubiquitylation and depletion of ATR and CHK1. Co-treatment with bortezomib, a proteasome inhibitor, restored PS-mediated depletion of ATR and CHK1. Notably, treatment with panobinostat induced hyperacetylation of both nuclear and cytoplasmic hsp90. This inhibited the chaperone association of nuclear hsp90 with ATR and CHK1, thereby promoting their degradation. PS treatment induced γ-H2AX levels, which were inhibited by co-treatment with N-acetylcysteine, suggesting that PS-induced ROS is involved in DNA damage. Moreover, PS treatment further increased α-irradiation induced DNA damage and its repair, characterized by the increase in α-irradiation-induced comet tail moment and the lack of DNA repair-mediated attenuation of the comet tail moment. To determine which class I HDAC is involved in deacetylation of nuclear hsp90, we individually knocked down (KD) HDAC 1, 2 and 3, by utilizing specific shRNA, and determined the effect on nuclear hsp90 hyperacetylation and the levels of ATR and CHK1. Our findings show that HDAC3 binds to hsp90 and KD of HDAC3, but not of HDAC1 or 2, caused hyperacetylation of nuclear hsp90 and depletion of ATR and CHK1. This was also observed in HDAC3 null mouse embryonic fibroblasts (MEFs). Ectopic overexpression of HDAC3 inhibited nuclear hsp90 acetylation in transformed cells. These findings demonstrate that HDAC3 is the nuclear hsp90 lysine deacetylase. They also demonstrate that genetic KD of HDAC3, or its inhibition by pan-HDAC inhibitors, induces hyperacetylation of hsp90, mediates loss of hsp90 chaperone association and depletion of ATR and CHK1, abrogates α-irradiation-induced DDR, and results in sensitization of transformed cells to DNA damage due to α-irradiation or replication stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4698. doi:1538-7445.AM2012-4698

Abstract 2119: DDB2 and XPC influence ATR and ATM recruitment and regulate DNA damage response upon UV irradiation

Abstract DNA damage response (DDR) pathways control the activation of DNA repair mechanisms, the activation and eventual release of cell cycle checkpoints to prevent replication of damaged DNA, and apoptosis as a last resort. These processes suggest the coordination of numerous proteins required to preserve genomic integrity. ATR and ATM kinases are central to checkpoint activation in response to DNA damage and replication stress. Activated ATR and ATM phosphorylate several proteins involved in DNA repair and cell cycle arrest. The nature of the signal, which activates ATR and ATM in response to UV damage, is not clear. Here, we showed that DDB2 and XPC, two early damage recognition factors, promote ATR and ATM recruitment and phosphorylation. ATR and ATM localize to the damage site and physically interact with XPC and DDB2. ATR and ATM recruitment and their phosphorylation are affected in cells defective in DDB2 and XPC function. In contrast, ATR- and ATM-deficiency do not regulate DDB2 and XPC recruitment to the damage site. Consequently, phosphorylation of ATR and ATM substrates Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutated cells, indicating that defective DDB2 and XPC function impair checkpoint signal transduction cascade in response to UV damage. Furthermore, DDB2 and XPC also regulate BRCA1 and Rad51 recruitment to the damage site, implicating their role in homologous recombination-mediated DNA repair pathway. Collectively, these data showed that ATR and ATM are present in the preincision complex with XPC, and influence DNA repair and checkpoint signaling pathways. In support of this, we revealed that depletion of ATR and ATM influence NER efficiency. These results are consistent with a model in which DDB2 and XPC cross-talk with ATR and ATM during maintenance of genomic integrity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2119. doi:1538-7445.AM2012-2119

Hsp90 Inhibitor–Mediated Disruption of Chaperone Association of ATR with Hsp90 Sensitizes Cancer Cells to DNA Damage

Following DNA damage that results in stalled replication fork, activation of ATR-CHK1 signaling induces the DNA damage response (DDR) in transformed cells. In the present studies on human cervical and breast cancer cells, we determined the effects of hsp90 inhibition on the levels and accumulation of DNA damage/repair-associated proteins following exposure to γ-ionizing radiation (IR; 4 Gy). We show that hsp90 inhibition with 17-allylamino-demehoxygeldanamycin or the novel, nongeldanamycin analogue AUY922 (resorcinylic isoxazole amide; Novartis Pharma) dose-dependently reduced the levels of ATR and CHK1 without affecting ATM levels. AUY922-mediated depletion of ATR and CHK1 was associated with an increase in their polyubiquitylation and decreased binding to hsp90. Cotreatment with bortezomib partially restored AUY922-mediated depletion of ATR and CHK1 levels. Additionally, treatment with AUY922 reduced the accumulation of ATR, p53BP1, and CHK1 but not γ-H2AX to the sites of DNA damage. Following exposure to IR, AUY922 treatment abrogated IR-induced phospho (p)-ATR and p-CHK1 levels, but significantly enhanced γ-H2AX levels. AUY922 treatment also increased IR-induced accumulation of the cells in G(2)-M phase of the cell cycle, inhibited the repair of IR-induced DNA damage, and augmented IR-mediated loss of clonogenic survival. Short hairpin RNA-mediated depletion of ATR also inhibited IR-induced p-ATR and p-CHK1, but increased γ-H2AX levels, sensitizing cancer cells to IR-induced apoptosis and loss of clonogenic survival. These findings indicate that ATR is a bona fide hsp90 client protein and post-IR administration of AUY922, by inhibiting ATR-CHK1-mediated DDR, sensitizes cancer cells to IR.

Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.

Inactivation of the Nijmegen Breakage Syndrome Gene Leads to Excess Centrosome Duplication via the ATR/BRCA1 Pathway

Nijmegen breakage syndrome is characterized by genomic instability and a predisposition for lymphoma and solid tumors. Nijmegen breakage syndrome 1 (NBS1), the protein which is mutated in these patients, functions in association with BRCA1 and ATR as part of the cellular response to DNA double-strand breaks. We show here that NBS1 forms foci at the centrosomes via an interaction with gamma-tubulin. Down-regulation of NBS1 by small interfering RNA induces supernumerary centrosomes, and this was confirmed with experiments using Nbs1 knockout mouse cells; the introduction of wild-type NBS1 (wt-NBS1) cDNA into these knockout mouse cells reduced the number of supernumerary centrosomes to normal levels. This phenotype in NBS1-deficient cells is caused by both centrosome duplication and impaired separation of centrioles, which have been observed in BRCA1-inhibited cells. In fact, supernumerary centrosomes were observed in Brca1 knockout mouse cells, and the frequency was not affected by NBS1 down-regulation, suggesting that NBS1 maintains centrosomes via a common pathway with BRCA1. This is consistent with findings that NBS1 physically interacts with BRCA1 at the centrosomes and is required for BRCA1-mediated ubiquitination of gamma-tubulin. Moreover, the ubiquitination of gamma-tubulin is compromised by either ATR depletion or an NBS1 mutation in the ATR interacting (FHA) domain, which is essential for ATR activation. These results suggest that, although centrosomes lack DNA, the NBS1/ATR/BRCA1 repair machinery affects centrosome behavior, and this might be a crucial role in the prevention of malignances.

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

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. 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. 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.