Genetic analysis of CHK1 and CHK2 homologues revealed a unique cross talk between ATM and ATR pathways in Neurospora crassa

DNA damage checkpoint is an important mechanism for organisms to maintain genome integrity. In Neurospora crassa, mus-9 and mus-21 are homologues of ATR and ATM, respectively, which are pivotal factors of DNA damage checkpoint in mammals. A N. crassa clock gene prd-4 has been identified as a CHK2 homologue, but its role in DNA damage response had not been elucidated. In this study, we identified another CHK2 homologue and one CHK1 homologue from the N. crassa genome database. As disruption of these genes affected mutagen tolerance, we named them mus-59 and mus-58, respectively. The mus-58 mutant was sensitive to hydroxyurea (HU), but the mus-59 and prd-4 mutants showed the same HU sensitivity as that of the wild-type strain. This indicates the possibility that MUS-58 is involved in replication checkpoint and stabilization of stalled forks like mammalian CHK1. Phosphorylation of MUS-58 and MUS-59 was observed in the wild-type strain in response to mutagen treatments. Genetic relationships between those three genes and mus-9 or mus-21 indicated that the mus-9 mutation was epistatic to mus-58, and mus-21 was epistatic to prd-4. These relationships correspond to two signal pathways, ATR-CHK1 and ATM-CHK2 that have been established in mammalian cells. However, both the mus-9 mus-59 and mus-21 mus-58 double mutants showed an intermediate level between the two parental strains for CPT sensitivity. Furthermore, these double mutants showed severe growth defects. Our findings suggest that the DNA damage checkpoint of N. crassa is controlled by unique mechanisms.     

Bleomycin-induced over-replication involves sustained inhibition of mitotic entry through the ATM/ATR pathway

Polyploid cells result in aneuploidy through aberrant chromosome segregation, possibly leading to tumorigenesis. Although polyploid cells are induced through over-replication by a variety of agents, including DNA-damaging drugs, the mechanisms that induce polyploidy have been hitherto unknown. Here, we show that treatment with bleomycin, a glycopeptide anticancer drug, induces over-replication at low cytotoxic doses. During bleomycin-induced over-replication, mitotic entry is inhibited through tyrosine phosphorylation of CDK1 along the ATM/ATR pathway in the early phase of treatment. Bleomycin-induced over-replication is inhibited by the inhibitors of the ATM/ATR pathway through abrogation of bleomycin-induced G2 arrest, and the ATM/ATR inhibitors promote cell death instead of over-replication. Following the phosphorylation of CDK1, the level of cyclin B1 is decreased in the late phase of treatment. Time-lapse imaging of clone cells that express a live cell marker of endogenous cyclin B1 revealed that cyclin B1 is degraded in G2-arrested cells upon bleomycin treatment. Our findings lead to a model of how the ATM/ATR pathway acts as a molecular switch for regulating cell fates, flipping between cell death via progress into mitosis, and over-replication via sustained G2 arrest upon DNA damage, where cyclin B1 degradation is an important factor for inducing over-replication.     

Interplays between ATM/Tel1 and ATR/Mec1 in sensing and signaling DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.     

ATM、ATR和DNA损伤介导的细胞周期阻滞

ATM和ATR属于PIKK家族,是DNA损伤检查点的主要成员。它们被不同类型的DNA损伤所激活,通过磷酸化相应的下游蛋白Chk1和Chk2等,调节细胞周期各个检查点,引起细胞周期阻滞,使DNA损伤得以修复。ATM和ATR在维持基因组的稳定性中起到至关重要的作用。本文着重综述有关ATM和ATR在DNA损伤介导的细胞周期阻滞中发挥的作用以及相互关系的最新研究进展。     

Homeostatic regulation of meiotic DSB formation by ATM/ATR

Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.     

MET inhibition enhances PARP inhibitor efficacy in castration-resistant prostate cancer by suppressing the ATM/ATR and PI3K/AKT pathways

Up to 30% of patients with metastatic castration-resistant prostate cancer (CRPC) patients carry altered DNA damage response genes, enabling the use of poly adenosine diphosphate–ribose polymerase (PARP) inhibitors in advanced CRPC. The proto-oncogene mesenchymal–epithelial transition (MET) is crucial in the migration, proliferation, and invasion of tumour cells. Aberrant expression of MET and its ligand hepatocyte growth factor is associated with drug resistance in cancer therapy. Here, we found that MET was highly expressed in human CRPC tissues and overexpressed in DU145 and PC3 cells in a drug concentration-dependent manner and is closely related to sensitivity to PARP inhibitors. Combining the PARP inhibitor olaparib with the MET inhibitor crizotinib synergistically inhibited CRPC cell growth both in vivo and in vitro. Further analysis of the underlying molecular mechanism underlying the MET suppression-induced drug sensitivity revealed that olaparib and crizotinib could together downregulate the ATM/ATR signaling pathway, inducing apoptosis by inhibiting the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, enhancing the olaparib-induced antitumour effect in DU145 and PC3 cells. In conclusion, we demonstrated that MET inhibition enhances sensitivity of CRPC to PARP inhibitors by suppressing the ATM/ATR and PI3K/AKT pathways and provides a novel, targeted therapy regimen for CRPC.     

ATR maintains select progenitors during nervous system development

The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder, ATR-Seckel syndrome. To understand how ATR functions during neurogenesis, we conditionally deleted Atr broadly throughout the murine nervous system, or in a restricted manner in the dorsal telencephalon. Unexpectedly, in both scenarios, Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest, other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs, in the nervous system, p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion, supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress, our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis.     

Both ATM and ATR promote the efficient and accurate processing of programmed meiotic double-strand breaks

The ATM and ATR protein kinases play central roles in the cellular response to double-strand breaks (DSBs) by regulating DNA repair, cell-cycle arrest and apoptosis. During meiosis, SPO11-dependent DSBs are generated, initiating recombination between homologous chromosomes. Previous studies in mice and plants have shown that defects in ATM result in the appearance of abnormally fragmented chromosomes. However, the role of ATR in promoting normal meiosis has not yet been elucidated. Employing null Arabidopsis mutants of ATR and ATM , we demonstrate here that although atr mutants display no obvious defects in any phase of meiotic progression, the combination of defects in atr and atm exacerbates the fragmentation observed in the atm single mutant, prevents complete synapsis of chromosomes, and results in extensive and persistent interactions between non-homologous DNAs. The observed non-homologous interactions require the induction of programmed breaks: the combination of either the atm single or the atr atm double mutant with a spo11 defect eliminates the ectopic interactions observed in the double mutant, as well as significantly reducing the fragmentation seen in atm or in atr atm . Our results suggest that ATM is required for the efficient processing of SPO11-dependent DSBs during meiosis. They also indicate that ATM and ATR act redundantly to inhibit sustained interactions between non-homologous chromatids, and that these ectopic interactions require SPO11 activity.     

ATM/ATR kinases link the synaptonemal complex and DNA double-strand break repair pathway choice

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Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.     

Regulation of Claspin degradation by the ubiquitin-proteosome pathway during the cell cycle and in response to ATR-dependent checkpoint activation

Claspin is involved in ATR-dependent activation of Chk1 during DNA replication and in response to DNA damage. We show that degradation of Claspin by the ubiquitin-proteosome pathway is regulated during the cell cycle. Claspin is stabilized in S-phase but is abruptly degraded in mitosis and is absent from early G(1) cells in which the phosphorylation of Chk1 by ATR is abrogated. In response to hydroxyurea, UV or aphidicolin, Claspin is phosphorylated in the Chk1-binding domain and its protein levels are increased in an ATR-dependent manner. Thus, the Chk1 pathway is regulated through both phosphorylation of Claspin and its controlled degradation.     

Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection

The protein kinases ataxia‐telangiectasia mutated (ATM) and ATM‐Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild‐type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.     

共济失调毛细血管扩张症突变基因与端粒不稳定性关系研究进展

在电离辐射等因素造成的DNA损伤修复信号传导过程中,共济失调毛细血管扩张症突变基因(ATM)起关键作用。同时,ATM属于P13K家族成员,其功能与保持端粒长度有关。端粒是真核细胞内染色体末端的重复的DNA序列,端粒的长短和稳定性决定了细胞的寿命。ATM突变导致端粒的不稳定性,包括端粒连接、端粒染色质结构变化,影响端粒聚集等。     

ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair

Ataxia telangiectasia (A-T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A-T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A-T pathogenesis. Here, we show that ATM promotes an anti-oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose-6-phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti-oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM-dependent PPP stimulation increases nucleotide production and that G6PD-deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs.     

Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis

The POT1 (protection of telomeres) protein binds the single-stranded G-rich overhang and is essential for both telomere end protection and telomere length regulation. Telomeric binding of POT1 is enhanced by its interaction with TPP1. In this study, we demonstrate that mouse Tpp1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres. Knockdown of Tpp1 elicits a p53-dependent growth arrest and an ATM-dependent DNA damage response at telomeres. In contrast to depletion of Trf2, which activates ATM, removal of Pot1a and Pot1b from telomeres initiates an ATR-dependent DNA damage response (DDR). Finally, we show that telomere dysfunction as a result of Tpp1 depletion promotes chromosomal instability and tumorigenesis in the absence of an ATM-dependent DDR. Our results uncover a novel ATR-dependent DDR at telomeres that is normally shielded by POT1 binding to the single-stranded G-overhang. In addition, our results suggest that loss of ATM can cooperate with dysfunctional telomeres to promote cellular transformation and tumor formation in vivo.     

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.