{"gene":"ASCC2","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2017,"finding":"ASCC2 contains a CUE domain that specifically recognizes K63-linked polyubiquitin chains, and this recognition is required for recruitment of the ASCC repair complex to nuclear foci upon alkylation damage. Loss of ASCC2 impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents but not other DNA damage types. RNF113A is identified as the upstream E3 ligase responsible for generating the K63-linked polyubiquitin signal.","method":"Nuclear foci imaging, CUE domain functional studies, KO/knockdown with alkylation sensitivity assays, epistasis with RNF113A","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — reciprocal localization, domain-specific mutants, KO with specific phenotypic readout, upstream E3 identified; replicated across multiple orthogonal approaches in single study","pmids":["29144457"],"is_preprint":false},{"year":2020,"finding":"ASCC2 is a component of the human RQC-trigger (hRQT) complex together with ASCC3 and TRIP4. The ubiquitin-binding activity of ASCC2 is required for triggering ribosome-associated quality control (RQC) in response to ribosome stalling, functioning analogously to yeast Cue3(Rqt3).","method":"Co-immunoprecipitation, dominant-negative mutants of ubiquitin-binding activity, ribosome stalling reporter assays, KD with RQC phenotype readout","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP defining complex composition, specific ubiquitin-binding mutants with functional RQC readout, ortholog mapping validated","pmids":["32099016"],"is_preprint":false},{"year":2019,"finding":"ASCC2 and ASCC3 bind to the ribosome and protect cells from toxic effects of selective ribosome-stalling compounds. Genetic interaction experiments place ASCC3 downstream of HBS1L and together with ASCC2 in the same pathway.","method":"Genome-wide CRISPRi screen, genetic interaction (epistasis) experiments, cell growth assays","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 — genome-wide screen with genetic epistasis; pathway placement supported but mechanistic detail limited to one study","pmids":["30875366"],"is_preprint":false},{"year":2021,"finding":"The ASCC2 CUE domain binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. Residues in the N-terminal portion of the ASCC2 α1 helix make unique contacts with the proximal ubiquitin, conferring K63-linkage specificity. Mutation of these residues decreases ASCC2 recruitment in response to DNA alkylation.","method":"Structural analysis (crystal/NMR), in vitro binding assays with diubiquitin, site-directed mutagenesis, cellular recruitment assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — structural determination combined with mutagenesis and functional validation in cells","pmids":["34971705"],"is_preprint":false},{"year":2020,"finding":"The structural basis for the ASCC2-ASCC3 interaction was determined: the ASCC3 fragment comprises a central helical domain and terminal extended arms that clasp the compact ASCC2 unit. Interfaces are evolutionarily conserved and harbor many somatic cancer mutation sites; cancer-associated mutations reduce ASCC2-ASCC3 binding affinity.","method":"Crystal structure of ASCC2-ASCC3 complex, quantitative binding assays, mapping of cancer mutations to interface","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional binding quantification and mutation analysis","pmids":["33139697"],"is_preprint":false},{"year":2018,"finding":"ASCC1 interacts with the ASCC complex via the ASCC3 helicase subunit and regulates proper recruitment of ASCC2 to alkylation damage foci. Loss of ASCC1 increases ASCC3 foci that lack ASCC2, indicating ASCC1 coordinates correct complex assembly. ASCC1 KO causes alkylation sensitivity epistatic with ASCC3.","method":"Co-immunoprecipitation, live-cell imaging of foci, CRISPR/Cas9 KO, epistasis analysis with alkylation sensitivity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, foci imaging, KO with specific phenotype; single lab study","pmids":["29997253"],"is_preprint":false},{"year":2026,"finding":"ASCC2 recruits ASCC3 to stalled replication forks. ASCC2's recruitment to stalled forks requires both its ubiquitin-binding activity and polyubiquitylation of PCNA at K164 catalyzed by SHPRH, HLTF, and RFWD3. ASCC3's DNA-unwinding activity downstream of ASCC2 promotes fork reversal, SMARCAL1 recruitment, RPA accumulation on ssDNA, and ATR activation.","method":"Co-IP, cellular recruitment assays with ubiquitin-binding mutants, in vitro DNA unwinding/fork reversal assays, epistasis with PCNA ubiquitylation pathway components","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro reconstitution of fork remodeling, mutant-specific recruitment failure, multiple orthogonal functional readouts in one study","pmids":["41785087"],"is_preprint":false},{"year":2026,"finding":"LncRNA DLEU1 promotes ASCC2 nuclear translocation and facilitates interaction between ASCC2 and ALKBH3 in gastric cancer cells, enhancing DNA repair and stabilizing E2F1 mRNA.","method":"RNA-protein interaction assays (RIP/pulldown), western blotting for nuclear fractionation, co-IP of ASCC2-ALKBH3 interaction","journal":"Biomarker research","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, pulldown and fractionation, partial mechanistic follow-up without full reconstitution","pmids":["41484982"],"is_preprint":false}],"current_model":"ASCC2 is a ubiquitin-binding scaffold subunit of the ASCC (and hRQT) complex whose CUE domain selectively recognizes K63-linked polyubiquitin (including at stalled replication forks via PCNA-K164 polyubiquitylation), thereby recruiting the ASCC3 helicase to sites of alkylation DNA damage and stalled ribosomes to drive ALKBH3-dependent dealkylation repair and ribosome-associated quality control, respectively; its structured interaction with ASCC3 (structurally defined by crystal analysis) is disrupted by somatic cancer mutations and regulated by the accessory subunit ASCC1."},"narrative":{"teleology":[{"year":2017,"claim":"The first mechanistic role for ASCC2 was established: its CUE domain selectively recognizes K63-linked polyubiquitin generated by RNF113A, and this recognition is required for ASCC complex recruitment to alkylation damage sites, resolving how the repair machinery is targeted to alkylated DNA.","evidence":"CUE domain mutagenesis, nuclear foci imaging, KO sensitivity assays with alkylating agents, epistasis with RNF113A in human cells","pmids":["29144457"],"confidence":"High","gaps":["Structural basis for K63-linkage selectivity of the CUE domain was not yet resolved","Whether ASCC2 functions beyond alkylation repair was unknown","The direct interaction interface between ASCC2 and ASCC3 had not been defined"]},{"year":2018,"claim":"ASCC1 was shown to regulate proper assembly of the ASCC complex at damage foci by ensuring ASCC2 co-localizes with ASCC3, establishing that an accessory subunit coordinates scaffold recruitment rather than ASCC2 acting autonomously.","evidence":"Co-IP, live-cell foci imaging, CRISPR KO with alkylation sensitivity epistasis in human cells","pmids":["29997253"],"confidence":"Medium","gaps":["ASCC1 interacts with ASCC3 rather than ASCC2 directly; the mechanism by which ASCC1 promotes ASCC2 recruitment is unclear","Single-lab study without independent replication"]},{"year":2019,"claim":"Genome-wide genetic screening extended ASCC2's function beyond DNA repair to ribosome stalling, showing that ASCC2 and ASCC3 protect cells from toxic ribosome-stalling compounds and operate in the same genetic pathway downstream of HBS1L.","evidence":"CRISPRi screen, genetic epistasis, cell growth assays in human cells","pmids":["30875366"],"confidence":"Medium","gaps":["Whether ASCC2's ubiquitin-binding activity was required for ribosome quality control was not tested","Biochemical mechanism of ribosome splitting was not addressed"]},{"year":2020,"claim":"Two advances converged: the crystal structure of the ASCC2–ASCC3 complex revealed the molecular interface (disrupted by cancer mutations), and ASCC2 was formally defined as a subunit of the human RQT complex whose ubiquitin-binding activity triggers ribosome-associated quality control upon ribosome stalling.","evidence":"Crystal structure with quantitative binding assays and cancer mutation mapping (ASCC2–ASCC3); Co-IP, dominant-negative ubiquitin-binding mutants, RQC reporter assays (hRQT complex)","pmids":["33139697","32099016"],"confidence":"High","gaps":["The ubiquitin signal on stalled ribosomes recognized by ASCC2 was not identified","No cryo-EM structure of the full hRQT-ribosome complex","The relevance of cancer mutations to tumor biology in vivo was not tested"]},{"year":2021,"claim":"The structural basis for K63-linkage specificity was resolved: unique contacts between the ASCC2 CUE domain α1 helix N-terminus and the proximal ubiquitin of K63-diubiquitin confer selectivity, and mutation of these residues ablates damage-induced recruitment.","evidence":"Crystal/NMR structure of CUE domain–K63-diubiquitin complex, in vitro binding assays, mutagenesis with cellular recruitment readouts","pmids":["34971705"],"confidence":"High","gaps":["How CUE domain engagement with polyubiquitin is coordinated with ASCC3 binding was not determined","No structural view of full-length ASCC2 in the context of the complete ASCC complex"]},{"year":2026,"claim":"ASCC2's ubiquitin-reading function was extended to replication stress: ASCC2 recruitment to stalled forks requires PCNA-K164 polyubiquitylation by SHPRH/HLTF/RFWD3, and ASCC3 helicase activity downstream promotes fork reversal, SMARCAL1 loading, and ATR activation.","evidence":"Co-IP, ubiquitin-binding mutant recruitment assays, in vitro fork reversal reconstitution, epistasis with PCNA ubiquitylation pathway","pmids":["41785087"],"confidence":"High","gaps":["Whether ASCC2 directly binds polyubiquitylated PCNA or an intermediate reader is involved was not resolved","In vivo relevance to replication-associated genome instability or tumor suppression not tested"]},{"year":null,"claim":"Key unresolved questions include how ASCC2 discriminates among its three recruitment contexts (alkylation damage, stalled ribosomes, stalled replication forks), whether competition or regulated switching occurs, and the identity of the ubiquitylated substrate recognized by ASCC2 at stalled ribosomes.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of ASCC2 in the context of a stalled ribosome or replication fork","The ubiquitin substrate at stalled ribosomes recognized by ASCC2 is unknown","Mechanism by which context-specific recruitment is regulated has not been addressed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,4,6]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[0,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,5,7]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,5,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[6]}],"complexes":["ASCC complex","hRQT complex"],"partners":["ASCC3","ASCC1","TRIP4","RNF113A","ALKBH3","PCNA"],"other_free_text":[]},"mechanistic_narrative":"ASCC2 is a ubiquitin-binding scaffold protein that couples K63-linked polyubiquitin recognition to the recruitment of the ASCC3 helicase at sites of alkylation DNA damage, stalled ribosomes, and stalled replication forks. Its CUE domain specifically contacts both the distal and proximal ubiquitin moieties of K63-linked diubiquitin, conferring linkage selectivity; this recognition is essential for nuclear foci formation upon alkylation damage (signaled by the E3 ligase RNF113A) and for triggering ribosome-associated quality control as part of the human RQT complex (with ASCC3 and TRIP4) [PMID:29144457, PMID:34971705, PMID:32099016]. Crystal structure analysis reveals that ASCC2 forms a compact unit clasped by ASCC3 helical and extended-arm segments, an interface disrupted by recurrent somatic cancer mutations, while the accessory subunit ASCC1 coordinates correct ASCC2 incorporation into the complex at damage foci [PMID:33139697, PMID:29997253]. At stalled replication forks, ASCC2 recruitment depends on PCNA-K164 polyubiquitylation by SHPRH/HLTF/RFWD3, and downstream ASCC3 helicase activity promotes fork reversal, SMARCAL1 recruitment, and ATR checkpoint activation [PMID:41785087]."},"prefetch_data":{"uniprot":{"accession":"Q9H1I8","full_name":"Activating signal cointegrator 1 complex subunit 2","aliases":["ASC-1 complex subunit p100","Trip4 complex subunit p100"],"length_aa":757,"mass_kda":86.4,"function":"Ubiquitin-binding protein involved in DNA repair and rescue of stalled ribosomes (PubMed:29144457, PubMed:32099016, PubMed:32579943, PubMed:36302773). Plays a role in DNA damage repair as component of the ASCC complex (PubMed:29144457). Recruits ASCC3 and ALKBH3 to sites of DNA damage by binding to polyubiquitinated proteins that have 'Lys-63'-linked polyubiquitin chains (PubMed:29144457). Part of the ASC-1 complex that enhances NF-kappa-B, SRF and AP1 transactivation (PubMed:12077347). Involved in activation of the ribosome quality control (RQC) pathway, a pathway that degrades nascent peptide chains during problematic translation (PubMed:32099016, PubMed:32579943, PubMed:36302773). Specifically recognizes and binds RPS20/uS10 ubiquitinated by ZNF598, promoting recruitment of the RQT (ribosome quality control trigger) complex on stalled ribosomes, followed by disassembly of stalled ribosomes (PubMed:36302773)","subcellular_location":"Nucleus; Nucleus speckle","url":"https://www.uniprot.org/uniprotkb/Q9H1I8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ASCC2","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DDX6","stoichiometry":0.2},{"gene":"DRG1","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2},{"gene":"RACK1","stoichiometry":0.2},{"gene":"RIOK3","stoichiometry":0.2},{"gene":"SRP68","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ASCC2","total_profiled":1310},"omim":[{"mim_id":"614217","title":"ACTIVATING SIGNAL COINTEGRATOR 1 COMPLEX, SUBUNIT 3; ASCC3","url":"https://www.omim.org/entry/614217"},{"mim_id":"614216","title":"ACTIVATING SIGNAL COINTEGRATOR 1 COMPLEX, SUBUNIT 2; ASCC2","url":"https://www.omim.org/entry/614216"},{"mim_id":"614215","title":"ACTIVATING SIGNAL COINTEGRATOR 1 COMPLEX, SUBUNIT 1; ASCC1","url":"https://www.omim.org/entry/614215"},{"mim_id":"604501","title":"THYROID HORMONE RECEPTOR INTERACTOR 4; TRIP4","url":"https://www.omim.org/entry/604501"},{"mim_id":"300951","title":"RING FINGER PROTEIN 113A; RNF113A","url":"https://www.omim.org/entry/300951"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Focal adhesion sites","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ASCC2"},"hgnc":{"alias_symbol":["ASC1p100","FLJ21588","DKFZp586O0223"],"prev_symbol":[]},"alphafold":{"accession":"Q9H1I8","domains":[{"cath_id":"-","chopping":"27-186","consensus_level":"high","plddt":94.968,"start":27,"end":186},{"cath_id":"-","chopping":"193-216_231-404","consensus_level":"high","plddt":93.5429,"start":193,"end":404},{"cath_id":"1.10.8.10","chopping":"466-524","consensus_level":"medium","plddt":81.6692,"start":466,"end":524}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1I8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1I8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1I8-F1-predicted_aligned_error_v6.png","plddt_mean":77.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ASCC2","jax_strain_url":"https://www.jax.org/strain/search?query=ASCC2"},"sequence":{"accession":"Q9H1I8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H1I8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H1I8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1I8"}},"corpus_meta":[{"pmid":"29144457","id":"PMC_29144457","title":"A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/29144457","citation_count":85,"is_preprint":false},{"pmid":"32099016","id":"PMC_32099016","title":"Identification of a novel trigger complex that facilitates ribosome-associated quality control in mammalian cells.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32099016","citation_count":82,"is_preprint":false},{"pmid":"30875366","id":"PMC_30875366","title":"Cellular response to small molecules that selectively stall protein synthesis by the ribosome.","date":"2019","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30875366","citation_count":27,"is_preprint":false},{"pmid":"30327447","id":"PMC_30327447","title":"Novel ASCC1 mutations causing prenatal-onset muscle weakness with arthrogryposis and congenital bone fractures.","date":"2018","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30327447","citation_count":27,"is_preprint":false},{"pmid":"29997253","id":"PMC_29997253","title":"RNA ligase-like domain in activating signal cointegrator 1 complex subunit 1 (ASCC1) regulates ASCC complex function during alkylation damage.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29997253","citation_count":26,"is_preprint":false},{"pmid":"33139697","id":"PMC_33139697","title":"The interaction of DNA repair factors ASCC2 and ASCC3 is affected by somatic cancer mutations.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33139697","citation_count":20,"is_preprint":false},{"pmid":"33777101","id":"PMC_33777101","title":"A Dual Systems Genetics Approach Identifies Common Genes, Networks, and Pathways for Type 1 and 2 Diabetes in Human Islets.","date":"2021","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33777101","citation_count":18,"is_preprint":false},{"pmid":"36819725","id":"PMC_36819725","title":"Sensogenomics of music and Alzheimer's disease: An interdisciplinary view from neuroscience, transcriptomics, and epigenomics.","date":"2023","source":"Frontiers in aging neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/36819725","citation_count":15,"is_preprint":false},{"pmid":"34971705","id":"PMC_34971705","title":"The ASCC2 CUE domain in the ALKBH3-ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin.","date":"2021","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/34971705","citation_count":8,"is_preprint":false},{"pmid":"36577525","id":"PMC_36577525","title":"Whole-genome characterization of myoepithelial carcinomas of the soft tissue.","date":"2022","source":"Cold Spring Harbor molecular case studies","url":"https://pubmed.ncbi.nlm.nih.gov/36577525","citation_count":8,"is_preprint":false},{"pmid":"35047834","id":"PMC_35047834","title":"Discovery of a neuromuscular syndrome caused by biallelic variants in ASCC3.","date":"2021","source":"HGG advances","url":"https://pubmed.ncbi.nlm.nih.gov/35047834","citation_count":8,"is_preprint":false},{"pmid":"40881169","id":"PMC_40881169","title":"Integrative multi-omics analysis and machine learning reveal the unique role of ASCC3 in combination with various immune-related genes in rectal adenocarcinoma.","date":"2025","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40881169","citation_count":2,"is_preprint":false},{"pmid":"41785087","id":"PMC_41785087","title":"Ski2-like helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses.","date":"2026","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/41785087","citation_count":1,"is_preprint":false},{"pmid":"40594069","id":"PMC_40594069","title":"Pan-cancer analysis reveals ASCC family promotes the cancer progression of lung adenocarcinoma.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40594069","citation_count":1,"is_preprint":false},{"pmid":"41484982","id":"PMC_41484982","title":"Histone modification-regulated LncRNA DLEU1 interacts with ASCC2/ALKBH3 complex to drive DNA repair, antioxidant homeostasis and glucose metabolism in gastric cancer.","date":"2026","source":"Biomarker research","url":"https://pubmed.ncbi.nlm.nih.gov/41484982","citation_count":0,"is_preprint":false},{"pmid":"40777259","id":"PMC_40777259","title":"The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40777259","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.19.629410","title":"Genome-wide association analyses in dairy heifers highlight genes overlapping with mouse and human fertility and human health traits","date":"2024-12-21","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.19.629410","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10454,"output_tokens":2034,"usd":0.030936},"stage2":{"model":"claude-opus-4-6","input_tokens":5310,"output_tokens":2235,"usd":0.123637},"total_usd":0.154573,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"ASCC2 contains a CUE domain that specifically recognizes K63-linked polyubiquitin chains, and this recognition is required for recruitment of the ASCC repair complex to nuclear foci upon alkylation damage. Loss of ASCC2 impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents but not other DNA damage types. RNF113A is identified as the upstream E3 ligase responsible for generating the K63-linked polyubiquitin signal.\",\n      \"method\": \"Nuclear foci imaging, CUE domain functional studies, KO/knockdown with alkylation sensitivity assays, epistasis with RNF113A\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal localization, domain-specific mutants, KO with specific phenotypic readout, upstream E3 identified; replicated across multiple orthogonal approaches in single study\",\n      \"pmids\": [\"29144457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ASCC2 is a component of the human RQC-trigger (hRQT) complex together with ASCC3 and TRIP4. The ubiquitin-binding activity of ASCC2 is required for triggering ribosome-associated quality control (RQC) in response to ribosome stalling, functioning analogously to yeast Cue3(Rqt3).\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative mutants of ubiquitin-binding activity, ribosome stalling reporter assays, KD with RQC phenotype readout\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP defining complex composition, specific ubiquitin-binding mutants with functional RQC readout, ortholog mapping validated\",\n      \"pmids\": [\"32099016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ASCC2 and ASCC3 bind to the ribosome and protect cells from toxic effects of selective ribosome-stalling compounds. Genetic interaction experiments place ASCC3 downstream of HBS1L and together with ASCC2 in the same pathway.\",\n      \"method\": \"Genome-wide CRISPRi screen, genetic interaction (epistasis) experiments, cell growth assays\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen with genetic epistasis; pathway placement supported but mechanistic detail limited to one study\",\n      \"pmids\": [\"30875366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The ASCC2 CUE domain binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. Residues in the N-terminal portion of the ASCC2 α1 helix make unique contacts with the proximal ubiquitin, conferring K63-linkage specificity. Mutation of these residues decreases ASCC2 recruitment in response to DNA alkylation.\",\n      \"method\": \"Structural analysis (crystal/NMR), in vitro binding assays with diubiquitin, site-directed mutagenesis, cellular recruitment assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural determination combined with mutagenesis and functional validation in cells\",\n      \"pmids\": [\"34971705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The structural basis for the ASCC2-ASCC3 interaction was determined: the ASCC3 fragment comprises a central helical domain and terminal extended arms that clasp the compact ASCC2 unit. Interfaces are evolutionarily conserved and harbor many somatic cancer mutation sites; cancer-associated mutations reduce ASCC2-ASCC3 binding affinity.\",\n      \"method\": \"Crystal structure of ASCC2-ASCC3 complex, quantitative binding assays, mapping of cancer mutations to interface\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional binding quantification and mutation analysis\",\n      \"pmids\": [\"33139697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ASCC1 interacts with the ASCC complex via the ASCC3 helicase subunit and regulates proper recruitment of ASCC2 to alkylation damage foci. Loss of ASCC1 increases ASCC3 foci that lack ASCC2, indicating ASCC1 coordinates correct complex assembly. ASCC1 KO causes alkylation sensitivity epistatic with ASCC3.\",\n      \"method\": \"Co-immunoprecipitation, live-cell imaging of foci, CRISPR/Cas9 KO, epistasis analysis with alkylation sensitivity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, foci imaging, KO with specific phenotype; single lab study\",\n      \"pmids\": [\"29997253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ASCC2 recruits ASCC3 to stalled replication forks. ASCC2's recruitment to stalled forks requires both its ubiquitin-binding activity and polyubiquitylation of PCNA at K164 catalyzed by SHPRH, HLTF, and RFWD3. ASCC3's DNA-unwinding activity downstream of ASCC2 promotes fork reversal, SMARCAL1 recruitment, RPA accumulation on ssDNA, and ATR activation.\",\n      \"method\": \"Co-IP, cellular recruitment assays with ubiquitin-binding mutants, in vitro DNA unwinding/fork reversal assays, epistasis with PCNA ubiquitylation pathway components\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro reconstitution of fork remodeling, mutant-specific recruitment failure, multiple orthogonal functional readouts in one study\",\n      \"pmids\": [\"41785087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"LncRNA DLEU1 promotes ASCC2 nuclear translocation and facilitates interaction between ASCC2 and ALKBH3 in gastric cancer cells, enhancing DNA repair and stabilizing E2F1 mRNA.\",\n      \"method\": \"RNA-protein interaction assays (RIP/pulldown), western blotting for nuclear fractionation, co-IP of ASCC2-ALKBH3 interaction\",\n      \"journal\": \"Biomarker research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, pulldown and fractionation, partial mechanistic follow-up without full reconstitution\",\n      \"pmids\": [\"41484982\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ASCC2 is a ubiquitin-binding scaffold subunit of the ASCC (and hRQT) complex whose CUE domain selectively recognizes K63-linked polyubiquitin (including at stalled replication forks via PCNA-K164 polyubiquitylation), thereby recruiting the ASCC3 helicase to sites of alkylation DNA damage and stalled ribosomes to drive ALKBH3-dependent dealkylation repair and ribosome-associated quality control, respectively; its structured interaction with ASCC3 (structurally defined by crystal analysis) is disrupted by somatic cancer mutations and regulated by the accessory subunit ASCC1.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ASCC2 is a ubiquitin-binding scaffold protein that couples K63-linked polyubiquitin recognition to the recruitment of the ASCC3 helicase at sites of alkylation DNA damage, stalled ribosomes, and stalled replication forks. Its CUE domain specifically contacts both the distal and proximal ubiquitin moieties of K63-linked diubiquitin, conferring linkage selectivity; this recognition is essential for nuclear foci formation upon alkylation damage (signaled by the E3 ligase RNF113A) and for triggering ribosome-associated quality control as part of the human RQT complex (with ASCC3 and TRIP4) [PMID:29144457, PMID:34971705, PMID:32099016]. Crystal structure analysis reveals that ASCC2 forms a compact unit clasped by ASCC3 helical and extended-arm segments, an interface disrupted by recurrent somatic cancer mutations, while the accessory subunit ASCC1 coordinates correct ASCC2 incorporation into the complex at damage foci [PMID:33139697, PMID:29997253]. At stalled replication forks, ASCC2 recruitment depends on PCNA-K164 polyubiquitylation by SHPRH/HLTF/RFWD3, and downstream ASCC3 helicase activity promotes fork reversal, SMARCAL1 recruitment, and ATR checkpoint activation [PMID:41785087].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"The first mechanistic role for ASCC2 was established: its CUE domain selectively recognizes K63-linked polyubiquitin generated by RNF113A, and this recognition is required for ASCC complex recruitment to alkylation damage sites, resolving how the repair machinery is targeted to alkylated DNA.\",\n      \"evidence\": \"CUE domain mutagenesis, nuclear foci imaging, KO sensitivity assays with alkylating agents, epistasis with RNF113A in human cells\",\n      \"pmids\": [\"29144457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for K63-linkage selectivity of the CUE domain was not yet resolved\",\n        \"Whether ASCC2 functions beyond alkylation repair was unknown\",\n        \"The direct interaction interface between ASCC2 and ASCC3 had not been defined\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"ASCC1 was shown to regulate proper assembly of the ASCC complex at damage foci by ensuring ASCC2 co-localizes with ASCC3, establishing that an accessory subunit coordinates scaffold recruitment rather than ASCC2 acting autonomously.\",\n      \"evidence\": \"Co-IP, live-cell foci imaging, CRISPR KO with alkylation sensitivity epistasis in human cells\",\n      \"pmids\": [\"29997253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"ASCC1 interacts with ASCC3 rather than ASCC2 directly; the mechanism by which ASCC1 promotes ASCC2 recruitment is unclear\",\n        \"Single-lab study without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genome-wide genetic screening extended ASCC2's function beyond DNA repair to ribosome stalling, showing that ASCC2 and ASCC3 protect cells from toxic ribosome-stalling compounds and operate in the same genetic pathway downstream of HBS1L.\",\n      \"evidence\": \"CRISPRi screen, genetic epistasis, cell growth assays in human cells\",\n      \"pmids\": [\"30875366\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether ASCC2's ubiquitin-binding activity was required for ribosome quality control was not tested\",\n        \"Biochemical mechanism of ribosome splitting was not addressed\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Two advances converged: the crystal structure of the ASCC2–ASCC3 complex revealed the molecular interface (disrupted by cancer mutations), and ASCC2 was formally defined as a subunit of the human RQT complex whose ubiquitin-binding activity triggers ribosome-associated quality control upon ribosome stalling.\",\n      \"evidence\": \"Crystal structure with quantitative binding assays and cancer mutation mapping (ASCC2–ASCC3); Co-IP, dominant-negative ubiquitin-binding mutants, RQC reporter assays (hRQT complex)\",\n      \"pmids\": [\"33139697\", \"32099016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The ubiquitin signal on stalled ribosomes recognized by ASCC2 was not identified\",\n        \"No cryo-EM structure of the full hRQT-ribosome complex\",\n        \"The relevance of cancer mutations to tumor biology in vivo was not tested\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The structural basis for K63-linkage specificity was resolved: unique contacts between the ASCC2 CUE domain α1 helix N-terminus and the proximal ubiquitin of K63-diubiquitin confer selectivity, and mutation of these residues ablates damage-induced recruitment.\",\n      \"evidence\": \"Crystal/NMR structure of CUE domain–K63-diubiquitin complex, in vitro binding assays, mutagenesis with cellular recruitment readouts\",\n      \"pmids\": [\"34971705\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How CUE domain engagement with polyubiquitin is coordinated with ASCC3 binding was not determined\",\n        \"No structural view of full-length ASCC2 in the context of the complete ASCC complex\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"ASCC2's ubiquitin-reading function was extended to replication stress: ASCC2 recruitment to stalled forks requires PCNA-K164 polyubiquitylation by SHPRH/HLTF/RFWD3, and ASCC3 helicase activity downstream promotes fork reversal, SMARCAL1 loading, and ATR activation.\",\n      \"evidence\": \"Co-IP, ubiquitin-binding mutant recruitment assays, in vitro fork reversal reconstitution, epistasis with PCNA ubiquitylation pathway\",\n      \"pmids\": [\"41785087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether ASCC2 directly binds polyubiquitylated PCNA or an intermediate reader is involved was not resolved\",\n        \"In vivo relevance to replication-associated genome instability or tumor suppression not tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how ASCC2 discriminates among its three recruitment contexts (alkylation damage, stalled ribosomes, stalled replication forks), whether competition or regulated switching occurs, and the identity of the ubiquitylated substrate recognized by ASCC2 at stalled ribosomes.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structure of ASCC2 in the context of a stalled ribosome or replication fork\",\n        \"The ubiquitin substrate at stalled ribosomes recognized by ASCC2 is unknown\",\n        \"Mechanism by which context-specific recruitment is regulated has not been addressed\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 4, 6]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 5, 7]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"ASCC complex\",\n      \"hRQT complex\"\n    ],\n    \"partners\": [\n      \"ASCC3\",\n      \"ASCC1\",\n      \"TRIP4\",\n      \"RNF113A\",\n      \"ALKBH3\",\n      \"PCNA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}