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Activation of the ATR kinase by the RPA-binding protein ETAA1

ABSTRACT: The ATR kinase is a master regulator of cellular responses to DNA damage and replication stress that is activated by a complex mechanism involving its binding partner ATRIP, RPA-coated ssDNA, the 9-1-1 complex and TopBP1. Here, we discovered a new ATR activation mechanism in human cells, mediated by the uncharacterized protein ETAA1 (Ewing’s tumor-associated antigen 1). From a comprehensive proteomic survey of protein recruitment to DNA double-strand break-modified chromatin, we identified ETAA1 as a factor that accumulates at DNA damage sites via an RPA-binding motif, and which has an important role in promoting cell survival and preventing replication fork breakage after genotoxic insults. Mechanistically, we show that ETAA1 harbors a conserved domain that potently and directly stimulates ATR kinase activity independent of TopBP1 and 9-1-1, and which is essential for the function of ETAA1 in the DNA damage response. Consistently, ablation of ETAA1 shows profound synthetic lethality with TopBP1 knockdown, resulting from massive replication fork collapse and abrogation of ATR-dependent signaling. Finally, we show that ETAA1 levels in cancer cell lines inversely correlate with their dependency on TopBP1 for ATR activation, and that overexpression of ETAA1 partially restores ATR-dependent signaling after loss of TopBP1. Together, these findings establish a new mechanism of ATR activation in human cells that operates in parallel with, but independent of, the canonical TopBP1-mediated pathway.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Xenopus Laevis (african Clawed Frog)

TISSUE(S): Egg, Cell Culture, Early Embryonic Cell

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD003529 | Pride | 2016-09-30

REPOSITORIES: Pride

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			Action	DRS
	20140905_QEp4_MaRa_SA_mr3p42_EXP02_HDC_020_01.raw	Raw
	20140905_QEp4_MaRa_SA_mr3p42_EXP02_HDC_020_02.raw	Raw
	20140905_QEp4_MaRa_SA_mr3p42_EXP02_HDC_020_03.raw	Raw
	20140905_QEp4_MaRa_SA_mr3p42_EXP02_HDC_040_01.raw	Raw
	20140905_QEp4_MaRa_SA_mr3p42_EXP02_HDC_040_02.raw	Raw

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Activation of the ATR kinase by the RPA-binding protein ETAA1.

Haahr Peter P Hoffmann Saskia S Tollenaere Maxim A X MA Ho Teresa T Toledo Luis Ignacio LI Mann Matthias M Bekker-Jensen Simon S Räschle Markus M Mailand Niels N

Nature cell biology 20161010 11

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fo ...[more]

PMID: 27723717

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Project description:Comprehensive Model Description Overview This model simulates the cellular response to DNA damage under conditions of ATM/ATR deficiency—such as in ataxia telangiectasia—and predicts the potential benefits of drug repositioning with Omaveloxolone and spermidine. It integrates several interconnected modules: DNA damage production and repair, ATM/ATR–p53 signaling, NRF2/KEAP1 regulation, spermidine-induced autophagy (with additional positive modulation of ATR signaling), and downstream p53 effectors involved in cell fate decisions. 1. DNA Damage Production and Repair Module Basal DNA Damage Production: DNA damage is continuously generated at a constant rate, representing both endogenous processes and low-level environmental stress. This reaction acts as a source term for the DNA_damage species. Repair Pathways: Several mechanisms work to reduce DNA damage: ATM-Mediated Repair: Active ATM facilitates the direct repair of DNA damage. ATR-Mediated Repair: Active ATR, in cooperation with CHK1, repairs DNA damage. NRF2-Mediated Repair: Active NRF2 triggers antioxidant responses that reduce DNA damage. Autophagy-Mediated Repair: The autophagy pathway, induced by spermidine, repairs DNA damage in a saturable manner (modeled using Michaelis–Menten kinetics). This ensures that even a significant pulse of damage is only partially repaired, leaving a nonzero steady-state level. 2. ATM/ATR–p53 Signaling Module ATM and ATR Activation: DNA damage, along with a positive input from TOPBP1, converts inactive ATM and ATR to their active forms. Under ATM/ATR-deficient conditions (as in ataxia telangiectasia), the activation is severely reduced, resulting in higher levels of unrepaired DNA damage. p53 Activation: Both ATM_active and ATR_active phosphorylate and activate p53. Active p53 then acts as a central integrator of the DNA damage signal. Regulatory Proteins: HDAC4: HDAC4 cycles between inactive and active states. Its active form can attenuate p53 activity. TOPBP1: TOPBP1 is activated in a DNA damage–dependent manner and helps promote ATR activation. CK2: CK2 is a constitutively active kinase that supports TOPBP1 activation. 3. NRF2/KEAP1 Module and Omaveloxolone NRF2 Activation: DNA damage stimulates the conversion of NRF2_inactive to NRF2_active. Active NRF2 enhances antioxidant defenses and promotes DNA repair. KEAP1-Mediated Degradation: Under normal conditions, KEAP1 targets NRF2_inactive for degradation, keeping NRF2 activity low. Modulation by Omaveloxolone: Omaveloxolone is modeled as a constant (boundary) species. It inhibits KEAP1’s activity, thereby reducing NRF2 degradation. This shifts the balance in favor of increased NRF2_active, which boosts the cell’s antioxidant and repair responses. 4. Spermidine-Induced Autophagy and Positive Modulation of ATR Signaling Spermidine is modeled as a constant species and has two major effects: Induction of Autophagy: Spermidine promotes the conversion of Autophagy_inactive to Autophagy_active. The active autophagy machinery repairs DNA damage using a saturable (Michaelis–Menten) mechanism. This pathway helps clear DNA damage but is limited by its maximum capacity. Enhancement of ATR Signaling: Spermidine also positively modulates ATR signaling by enhancing the CK2-mediated activation of TOPBP1. In the model, the effective rate of TOPBP1 activation is increased proportionally to the concentration of spermidine. This is represented by a modulation factor, where the effective rate constant for TOPBP1 activation is multiplied by a term that increases with spermidine concentration. The boosted TOPBP1_active level in turn enhances ATR activation, partially compensating for the loss of ATM/ATR function. 5. Downstream p53 Targets: Cell Fate Decisions Activated p53 regulates cell fate by inducing the production of several downstream effectors: p21: p21 is produced in response to p53 activation and is associated with cell cycle arrest and senescence. Its production is balanced by a degradation reaction. BAX and PUMA: Both BAX and PUMA are pro-apoptotic proteins. They are produced in proportion to p53_active and are subsequently degraded. Their relative levels help determine whether the cell will undergo apoptosis. 6. Reaction Kinetics (Qualitative Overview) Mass-Action and Michaelis–Menten Kinetics: Most reactions in the model are governed by mass-action kinetics. For example, activation and deactivation reactions (such as ATM activation by DNA damage or p53 activation by ATM/ATR) follow simple proportional relationships. Saturable Repair Processes: The autophagy-mediated DNA repair reaction uses Michaelis–Menten kinetics to reflect a saturation effect; when DNA damage is high, the repair rate approaches a maximum value, ensuring that not all damage is cleared immediately. Modulatory Effects: Spermidine increases the effective rate of TOPBP1 activation (and hence ATR activation) via a modulation term that is proportional to its concentration. Omaveloxolone reduces the degradation rate of NRF2 by inhibiting KEAP1. Biological Implications and Model Utility ATM/ATR Deficiency: In ataxia telangiectasia, ATM/ATR functions are diminished, leading to increased DNA damage. The model simulates this scenario and examines how alternative pathways (NRF2-mediated repair, autophagy, and enhanced ATR signaling via spermidine) help mitigate the damage. Drug Repositioning Predictions: The model can be used to predict the outcomes of repositioning drugs: Omaveloxolone is expected to stabilize NRF2, enhancing antioxidant defenses and reducing DNA damage. Spermidine acts dually by promoting autophagy (with a saturable repair capacity) and by boosting ATR signaling via TOPBP1 activation. Cell Fate Outcomes: The downstream p53 targets (p21, BAX, and PUMA) serve as markers for cell cycle arrest/senescence and apoptosis. By simulating the dynamics of these proteins, the model can predict whether a cell is likely to survive, arrest its cycle, or undergo apoptosis based on the integrated response to DNA damage and drug interventions. Conclusion This detailed and comprehensive model integrates multiple layers of the cellular response to DNA damage in ATM/ATR-deficient conditions. It includes upstream damage sensing, multiple repair pathways (including direct kinase-mediated repair, antioxidant responses via NRF2, and autophagy-mediated repair), and downstream effectors that determine cell fate. Omaveloxolone and spermidine are modeled to specifically modulate the NRF2/KEAP1 pathway and ATR signaling (via TOPBP1 activation), respectively. This framework provides a robust platform for exploring drug repositioning strategies and predicting cellular outcomes in diseases such as ataxia telangiectasia.