Affinage

ETAA1

Ewing's tumor-associated antigen 1 · UniProt Q9NY74

Length
926 aa
Mass
103.4 kDa
Annotated
2026-06-09
13 papers in source corpus 10 papers cited in narrative 10 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ETAA1 is a genome-maintenance factor that operates as an independent activator of the ATR-ATRIP checkpoint kinase, functioning in parallel to the TopBP1/9-1-1 pathway (PMID:27723717, PMID:27723720). It is recruited to RPA-coated single-stranded DNA at stalled replication forks through two conserved RPA-interaction motifs, where it engages the ATR-ATRIP complex via a conserved N-terminal ATR-activation domain (AAD) and directly stimulates ATR kinase activity; both RPA binding and an intact AAD are required for its function at forks (PMID:27723717, PMID:27818175). The AAD contains a coiled-coil motif that mediates direct ATR binding, a mechanism shared with the TopBP1 AAD (PMID:30940728), and ETAA1-driven ATR activation is strongly enhanced when ETAA1 is bound to RPA-ssDNA, defining RPA-ssDNA as a direct positive effector in a reconstituted system (PMID:30975033). The combined loss of ETAA1 and TopBP1 is synthetically lethal with massive genome instability, underscoring its non-redundant contribution to ATR signalling (PMID:27723717). Functional partitioning distinguishes the two activators: ETAA1 predominantly drives mitotic ATR signalling, sustaining Aurora B activity, proper chromosome alignment, and spindle assembly checkpoint function, whereas TopBP1 is the principal activator during replication stress (PMID:30755469). ETAA1's activity is gated by cell-cycle- and replication-stress-dependent phosphorylation of conserved AAD residues, which are required to prevent mitotic chromosome abnormalities, and becomes essential when replication fidelity is compromised (PMID:31615875). p130RB2 acts as a positive regulator of this axis, supporting the RPA32-ETAA1 interaction and ATR activation under replication stress (PMID:37201767). In mice, ETAA1 deficiency selectively impairs clonal expansion of effector T cells in a cell-autonomous manner associated with markers of replication stress, linking its ATR-activating role to proliferative demand (PMID:28607084).

Mechanistic history

Synthesis pass · year-by-year structured walk · 7 steps
  1. 2016 High

    Established that ATR activation does not depend solely on TopBP1, answering whether a parallel activator exists by identifying ETAA1 as an RPA-recruited, AAD-containing protein that directly stimulates ATR kinase activity.

    Evidence Co-IP, in vitro ATR kinase assays, domain mapping, loss-of-function and synthetic lethality analysis in human cells

    PMID:27723717 PMID:27723720 PMID:27818175

    Open questions at the time
    • No structure of the ETAA1 AAD-ATR/ATRIP interface
    • How ETAA1 versus TopBP1 usage is partitioned at individual lesions not resolved
    • Stoichiometry of RPA-ETAA1-ATR complex unknown
  2. 2019 High

    Defined the molecular basis of ATR binding by showing the AAD coiled-coil motif mediates direct ATR engagement, a mechanism shared with TopBP1.

    Evidence Bioinformatic prediction, in vitro kinase assays, Co-IP and mutagenesis

    PMID:30940728

    Open questions at the time
    • No high-resolution structure of the coiled-coil/ATR contact
    • Whether the coiled-coil engages the same ATR surface as TopBP1 not directly shown
  3. 2019 High

    Demonstrated that RPA-ssDNA is a direct positive effector of ETAA1-mediated ATR activation, not merely a recruitment platform, via reconstitution with defined components.

    Evidence In vitro reconstitution with recombinant full-length ETAA1, Xenopus egg-extract system, ATR-ATRIP kinase assay

    PMID:30975033

    Open questions at the time
    • Conformational mechanism by which RPA-ssDNA enhances AAD activity unknown
    • Quantitative contribution of RPA-ssDNA stimulation in cells not established
  4. 2019 High

    Distinguished the cellular division of labor between activators, showing ETAA1 predominantly drives mitotic ATR signalling required for Aurora B activity, chromosome alignment, and the spindle assembly checkpoint.

    Evidence Quantitative phosphoproteomics, ETAA1/TOPBP1 knockout/knockdown cells, Aurora B activity assay, live-cell imaging, SAC assays

    PMID:30755469

    Open questions at the time
    • Mechanism linking mitotic ATR signalling to Aurora B activation not defined
    • What recruits ETAA1 during mitosis when replication forks are absent unclear
  5. 2019 High

    Showed ETAA1 activity is regulated by cell-cycle- and stress-dependent AAD phosphorylation and is conditionally essential, answering how its activator function is tuned to replication demand.

    Evidence CRISPR-Cas9 genome-scale screen, phospho-site mutagenesis, chromosome stability assays

    PMID:31615875

    Open questions at the time
    • Identity of the kinase(s) phosphorylating the AAD not established
    • Mechanism by which phosphorylation modulates ATR binding/activation unknown
  6. 2017 Medium

    Linked ETAA1's ATR-activating function to a physiological proliferative process by showing its deficiency cell-autonomously impairs effector T cell clonal expansion with replication-stress markers.

    Evidence Forward genetic screen in mice, mutant breeding, in vivo infection/immunization, cell-autonomous transfer, H2AX/p53-target readouts

    PMID:28607084

    Open questions at the time
    • Pathway placement inferred from H2AX/p53 marker correlation rather than direct ATR-pathway rescue
    • Whether the T cell phenotype reflects mitotic versus replication-stress ATR signalling not distinguished
  7. 2023 Medium

    Identified p130RB2 as a positive co-regulator required for the RPA32-ETAA1 interaction and downstream ATR activation under replication stress.

    Evidence Reciprocal Co-IP, siRNA knockdown, ATR substrate phosphorylation assays, cell cycle and anaphase bridge analysis, rescue experiments

    PMID:37201767

    Open questions at the time
    • Single-lab study; reciprocal validation in other systems not yet performed
    • Whether p130RB2 acts structurally or via modification of ETAA1 unknown

Open questions

Synthesis pass · forward-looking unresolved questions
  • How phosphorylation of the AAD, RPA-ssDNA binding, and co-regulators such as p130RB2 are integrated to selectively license ETAA1 for mitotic versus replication-stress ATR signalling remains unresolved.
  • No structural model of the active ETAA1-ATR-ATRIP complex
  • Upstream kinase(s) controlling AAD phosphorylation unidentified
  • Mechanistic basis for mitotic specificity not established

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 4 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005634 nucleus 2
Pathway
R-HSA-73894 DNA Repair 3 R-HSA-1640170 Cell Cycle 2 R-HSA-8953897 Cellular responses to stimuli 2
Partners
ATRATRIPBLMRBL2RMI1RMI2RPA32TOP3A

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2016 ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and contains a conserved ATR-activation domain (AAD) that directly and potently stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 causes synthetic lethality with massive genome instability and abrogation of ATR-dependent signalling, establishing ETAA1 as an independent, parallel ATR activator. Co-immunoprecipitation, in vitro kinase assays, loss-of-function (siRNA/genetic depletion), immunofluorescence, synthetic lethality analysis Nature cell biology High 27723717
2016 ETAA1 contains two RPA-interaction motifs that localize it to stalled replication forks, and a motif with sequence similarity to the TOPBP1 ATR-activation domain that directly binds ATR/ATRIP and activates ATR. ETAA1 also interacts with the BLM/TOP3α/RMI1/RMI2 complex and the ATR/ATRIP complex. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway. Proteomic screens, co-immunoprecipitation, pulldown, in vitro ATR kinase assay, RPA-interaction domain mapping, ETAA1-deficient cell lines with genome instability readouts Nature cell biology High 27723720
2016 ETAA1 interacts with RPA via two conserved RPA-binding domains and is recruited to stalled replication forks, where it activates ATR through a conserved N-terminal ATR-activating domain (AAD). Both RPA binding and ATR activation are required for ETAA1 function at stalled forks. Co-immunoprecipitation, immunofluorescence, domain-deletion/mutation analysis, ATR signalling assays in ETAA1-depleted cells Current biology : CB High 27818175
2019 Both ETAA1 and TOPBP1 AADs contain a predicted coiled-coil motif required for ATR activation in vitro and in cells. Mutation of the coiled-coil does not alter AAD oligomerization but impairs binding of the AADs to ATR, indicating that the coiled-coil mediates ATR binding as a shared activation mechanism. Bioinformatic analysis, in vitro kinase assays, co-immunoprecipitation, immunofluorescence-based signalling assays, mutagenesis The Journal of biological chemistry High 30940728
2019 RPA-coated single-stranded DNA (ssDNA) greatly stimulates ETAA1-mediated activation of ATR-ATRIP. In a defined in vitro reconstitution system, full-length recombinant ETAA1 activates ATR-ATRIP, and this activity is strongly enhanced when ETAA1 is bound to RPA on ssDNA, establishing RPA-ssDNA as a direct positive effector of ETAA1-mediated ATR activation. In vitro reconstitution with recombinant proteins, Xenopus egg-extract system, ATR-ATRIP kinase assay with defined components Cell cycle (Georgetown, Tex.) High 30975033
2019 Quantitative phosphoproteomics revealed that ETAA1 predominantly regulates mitotic ATR signalling, while TOPBP1 is the primary ATR activator for replication stress. Inactivation of ATR or ETAA1 (but not TOPBP1) decreases Aurora B kinase activity during mitosis. ETAA1-mediated ATR activation is required for proper chromosome alignment in metaphase and a fully functional spindle assembly checkpoint. Quantitative mass spectrometry phosphoproteomics, ETAA1/TOPBP1 knockout/knockdown cells, Aurora B activity assay, live-cell imaging of chromosome alignment, spindle assembly checkpoint assay The Journal of cell biology High 30755469
2019 The ATR-activating potential of ETAA1 is controlled by cell cycle- and replication stress-dependent phosphorylation of conserved residues within its AAD. These stimulatory phosphorylations are required for ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. A CRISPR-Cas9 genome-scale screen confirmed ETAA1's ATR-stimulating function becomes indispensable when DNA replication fidelity is compromised. CRISPR-Cas9 genome-scale screen, phosphorylation-site mutagenesis, cell cycle and replication stress-dependent phosphorylation analysis, chromosome stability assays The Journal of cell biology High 31615875
2017 In mice, ETAA1 deficiency (via exon-2-skipping or truncating alleles) selectively impairs clonal expansion of effector CD8+ and CD4+ T cells following infection/immunization without affecting immune cell development. The defect is cell-autonomous and is associated with increased TP53-induced mRNAs and phosphorylation of H2AX (markers of ATR/ATM-mediated replication stress), linking ETAA1's ATR-activating function (encoded in part by exon 2) to T cell proliferation. Forward genetic screen in mice, homozygous mutant breeding, in vivo viral infection/immunization models, cell-autonomous transfer assays, H2AX phosphorylation and p53-target gene expression readouts Proceedings of the National Academy of Sciences of the United States of America Medium 28607084
2023 p130RB2 binds ETAA1 (but not TopBP1) and is required for the RPA32-ETAA1 interaction under hydroxyurea-induced replication stress. Depletion of p130RB2 reduces ATR activation (phosphorylation of RPA32, Chk1, and ATR itself) and impairs proper S-phase re-progression, establishing p130RB2 as a positive regulator of the RPA32-ETAA1-ATR axis. Co-immunoprecipitation, siRNA knockdown, ATR substrate phosphorylation assays, cell cycle analysis, anaphase bridge quantification, rescue experiments Biochimica et biophysica acta. Molecular cell research Medium 37201767
2005 ETAA1 (ETAA16) encodes a 926-amino-acid protein (predicted MW ~103 kDa) whose epitope recognized by antibody Ak16 maps to its central region, which is part of an extracellular domain. Cell surface expression detected by flow cytometry is restricted to Ewing's tumour cell lines. The gene locus was mapped to chromosome 2p13-15 by FISH. cDNA library immunoscreening, flow cytometry, immunohistochemistry, RT-PCR, FISH Cancer immunology, immunotherapy : CII Low 16003559

Source papers

Stage 0 corpus · 13 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2016 Activation of the ATR kinase by the RPA-binding protein ETAA1. Nature cell biology 202 27723717
2016 ETAA1 acts at stalled replication forks to maintain genome integrity. Nature cell biology 200 27723720
2016 RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response. Current biology : CB 112 27818175
2019 Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1. The Journal of cell biology 52 30755469
2019 Common motifs in ETAA1 and TOPBP1 required for ATR kinase activation. The Journal of biological chemistry 33 30940728
2019 Regulation of ETAA1-mediated ATR activation couples DNA replication fidelity and genome stability. The Journal of cell biology 17 31615875
2017 Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion. Proceedings of the National Academy of Sciences of the United States of America 16 28607084
2019 RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR. Cell cycle (Georgetown, Tex.) 15 30975033
2005 Structure and function of ETAA16: a novel cell surface antigen in Ewing's tumours. Cancer immunology, immunotherapy : CII 13 16003559
2022 Genetic polymorphisms within the ETAA1 gene associated with growth traits in Chinese sheep breeds. Animal genetics 9 35352359
2019 ETAA1 ensures proper chromosome segregation: A matter of S phase or mitosis? The Journal of cell biology 3 31685532
2024 Exploring RPA1-ETAA1 axis via high-throughput data analysis: implications for PD-L1 nuclear translocation and tumor-immune dynamics in liver cancer. Frontiers in immunology 1 39660144
2023 p130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis. Biochimica et biophysica acta. Molecular cell research 0 37201767

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