{"gene":"CDC16","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1995,"finding":"CDC16 (human homolog of S. cerevisiae Cdc16) is a subunit of the 20S anaphase-promoting complex (APC), a mitosis-specific ubiquitin-protein ligase that catalyzes conjugation of ubiquitin to cyclin B, targeting it for proteasomal degradation at the onset of anaphase. Immunopurified CDC27-containing complexes (which include CDC16) are sufficient to reconstitute cyclin B ubiquitination activity.","method":"Biochemical fractionation of mitotic Xenopus egg extracts, immunodepletion with CDC27 antibodies, immunopurification, in vitro ubiquitination reconstitution assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro ubiquitin ligase activity, foundational study replicated widely","pmids":["7736580"],"is_preprint":false},{"year":1995,"finding":"Human CDC16Hs (encoded by a cDNA homologous to S. cerevisiae CDC16) colocalizes with CDC27Hs to the centrosome throughout the mammalian cell cycle and to the mitotic spindle. Microinjection of anti-CDC27Hs antibodies arrests HeLa cells in metaphase, demonstrating that the CDC16/CDC27-containing complex is essential for the metaphase-to-anaphase transition.","method":"Immunofluorescence microscopy, antibody microinjection into HeLa cells, cell cycle arrest assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — direct localization experiment with functional (arrest) consequence, replicated in complementary study","pmids":["7736578"],"is_preprint":false},{"year":1994,"finding":"The fission yeast cut9+ gene (encoding the S. pombe ortholog of Cdc16/APC6) is required for the initiation of anaphase; temperature-sensitive cut9 mutants enter mitosis and form short spindles but block at anaphase onset, while postanaphase events (spindle disassembly, septation, cytokinesis) continue inappropriately. Cut9 is a 78-kDa TPR-containing protein essential for viability.","method":"Temperature-sensitive mutant analysis, genetic epistasis (suppressor genes nuc2+, scn1, scn2), microscopy of cell cycle progression","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis and phenotypic analysis in a clear ortholog, strong evidence","pmids":["7798319"],"is_preprint":false},{"year":1996,"finding":"CDC16 and CDC27 in S. cerevisiae are required to restrict DNA replication to once per cell cycle; conditional cdc16 mutants rereplicate all chromosomes (up to 8C DNA content) within a single cell cycle without passing through mitosis, despite persistently elevated Clb2-Cdc28 (CDK) kinase activity, indicating CDC16 prevents rereplication independently of CDK-mediated inhibition of re-initiation.","method":"Conditional mutant analysis (temperature-sensitive alleles), flow cytometry (DNA content), genetic screen","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — clean loss-of-function with defined molecular phenotype, mechanistic follow-up","pmids":["8620535"],"is_preprint":false},{"year":1997,"finding":"The serine/threonine phosphatase PP5 physically interacts with CDC16 and CDC27, two TPR-containing subunits of the APC/C. The interaction requires only the N-terminal TPR-containing domain of PP5, while the binding site on CDC16 and CDC27 maps to their C-terminal TPR block. PP5 colocalizes with CDC16/CDC27 at the mitotic spindle apparatus, suggesting it may regulate APC/C activity through dephosphorylation.","method":"Yeast two-hybrid analysis, in vitro binding assay, deletion mapping, indirect immunofluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — two-hybrid plus in vitro binding with localization data, single lab","pmids":["9405394"],"is_preprint":false},{"year":1998,"finding":"In S. cerevisiae cdc16 mutants, rereplication initiates specifically from normal chromosomal replication origins, and requires both the initiation proteins Cdc6p and Cdc46/Mcm5p — proteins thought to be inactivated by CDK. This establishes that CDC16 is required to prevent inappropriate firing of replication origins after S-phase, acting independently of CDK-mediated origin suppression.","method":"Two-dimensional DNA gel electrophoresis, density-transfer experiments, genetic epistasis (cdc6 and cdc46 double mutants)","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (2D gels, density transfer, epistasis) establishing mechanistic pathway","pmids":["9660930"],"is_preprint":false},{"year":2003,"finding":"The Arabidopsis NOMEGA gene encodes the plant ortholog of APC6/CDC16; nomega mutant embryo sacs arrest at the two-nucleate stage and are unable to degrade Cyclin B, directly demonstrating that the CDC16-containing APC/C is required for Cyclin B proteolysis during cell cycle progression in plant gametophyte development.","method":"Genetic mutant characterization, cyclin B degradation assay in vivo, complementation","journal":"The Plant journal","confidence":"Medium","confidence_rationale":"Tier 2 — functional assay (cyclin B stability) in loss-of-function mutant, ortholog context","pmids":["14675450"],"is_preprint":false},{"year":2004,"finding":"Swm1/Apc13 is an evolutionarily conserved APC/C subunit that promotes stable association of CDC16 and CDC27 with the complex; deletion of SWM1 reduces the incorporation of Cdc16 and Cdc27 into the APC/C, abolishes ubiquitin ligase activity in vitro, and delays APC/C-dependent cell cycle events in vivo.","method":"Affinity purification, mass spectrometry, in vitro ubiquitin ligase activity assay, in vivo cell cycle analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (AP-MS, in vitro ligase assay, in vivo phenotype) establishing subunit interaction requirement","pmids":["15060174"],"is_preprint":false},{"year":2003,"finding":"At least 34 mitosis-specific phosphorylation sites exist on human APC/C subunits; 32 of these cluster in Apc1 and TPR subunits including CDC16 (Cdc16), CDC27, CDC23, and Apc7. Cdk1 can generate at least 15 mitotic phospho-sites in vitro. APC phosphorylation by Cdk1 (but not Plk1) is sufficient for increased CDC20 binding and APC activation. Phospho-APC accumulates at centrosomes in prometaphase where cyclin B ubiquitination initiates.","method":"Mass spectrometry phospho-site mapping, in vitro kinase assays (Cdk1, Plk1), co-immunoprecipitation, immunofluorescence with phospho-specific antibodies","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — phospho-site mapping combined with in vitro kinase assays and localization, multiple methods","pmids":["14657031"],"is_preprint":false},{"year":2010,"finding":"The crystal structure of S. pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26) reveals that Cdc16/Cut9 is a contiguous superhelix of 14 TPR units. The C-terminal TPR block interacts with Hcn1/Cdc26, while the N-terminal TPR block mediates CDC16 self-association through a homotypic dimer interface structurally related to the Cdc27 dimerization domain. The acetylated N-terminal Met of Hcn1 is enclosed within the Cut9 TPR chamber, protecting Hcn1 from Doa10-mediated ubiquitin-dependent degradation.","method":"X-ray crystallography, biochemical characterization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with functional interpretation and mechanistic validation","pmids":["20924356"],"is_preprint":false},{"year":2009,"finding":"Biophysical and structural studies show that CDC26 stabilizes the structure of APC6 (CDC16) through an intermolecular TPR mimic composed of one helix from each protein; genetic studies confirm that this interaction is required for APC integrity.","method":"X-ray crystallography, biophysical binding assays, genetic complementation","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with genetic validation, mechanistic explanation for CDC26 function","pmids":["19668213"],"is_preprint":false},{"year":2022,"finding":"DEPDC1B competitively associates with human CDC16 (an APC/C ubiquitin ligase subunit) to sequester it, thereby preventing CDC16-mediated ubiquitination and proteasomal degradation of the secreted protein SCUBE3. This stabilization of SCUBE3 promotes melanoma angiogenesis and metastasis.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression rescue, in vivo tumor/angiogenesis models","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP plus ubiquitination assay and in vivo models, single lab","pmids":["35088579"],"is_preprint":false},{"year":2018,"finding":"YDJC physically interacts with CDC16 as shown by co-immunoprecipitation; CDC16 siRNA knockdown induces sphingosylphosphorylcholine-triggered keratin reorganization, migration, and invasion in A549 lung cancer cells, while CDC16 overexpression blocks these events, suggesting CDC16 suppresses EMT-related signaling.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, migration/invasion assays","journal":"Oncotarget","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP plus knockdown/overexpression phenotype, no direct mechanistic pathway placement","pmids":["29796162"],"is_preprint":false},{"year":2019,"finding":"CDC16 overexpression induces ubiquitination of YDJC protein and increases PP2A expression; YDJC overexpression escapes CDC16-mediated ubiquitination (dependent on YDJC deacetylase activity) and promotes PP2A ubiquitination, leading to EMT in lung cancer cells. This places CDC16 as a ubiquitin ligase regulator in a YDJC-PP2A-ERK2-EMT axis.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression with deacetylase-dead mutant (YDJCD13A), in vivo orthotopic mouse model","journal":"Journal of oncology","confidence":"Low","confidence_rationale":"Tier 3 — mechanistic claims based on overexpression and co-IP, single lab, limited controls","pmids":["31485224"],"is_preprint":false},{"year":2025,"finding":"c-Jun competitively interacts with CDC16, a key APC/C subunit, reducing APC complex formation and inhibiting cell cycle progression from G1 in neuroblastoma cells. c-Jun overexpression inhibits neuroblastoma cell proliferation and migration via sequestration of CDC16.","method":"Co-immunoprecipitation, flow cytometry (cell cycle), EdU proliferation assay, transwell migration assay, retinoic acid treatment","journal":"Biology direct","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP with cellular phenotype assays, single lab, limited mechanistic depth","pmids":["40149013"],"is_preprint":false}],"current_model":"CDC16/APC6 is a TPR-repeat scaffold subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase: it forms a 14-TPR superhelix that homo-dimerizes via its N-terminal TPR block, binds CDC26 via an intermolecular TPR mimic to stabilize APC/C integrity, and is phosphorylated by Cdk1 at mitosis-specific sites to enable APC/C activation and CDC20 recruitment, collectively driving ubiquitination of cyclin B and other substrates to trigger anaphase onset and prevent DNA re-replication."},"narrative":{"teleology":[{"year":1994,"claim":"Identification of the fission yeast ortholog Cut9 as a TPR protein essential for anaphase initiation established that CDC16-family genes are required for the metaphase-to-anaphase transition.","evidence":"Temperature-sensitive cut9 mutants in S. pombe arrest at anaphase onset; genetic epistasis with nuc2+, scn1, scn2","pmids":["7798319"],"confidence":"High","gaps":["Biochemical function of Cut9/CDC16 unknown","No direct connection to ubiquitin-dependent proteolysis at this stage"]},{"year":1995,"claim":"Biochemical purification of the APC/C as a cyclin B ubiquitin ligase containing CDC16 and localization of CDC16 to the centrosome and mitotic spindle established CDC16 as a core subunit of the mitosis-specific E3 ligase driving anaphase.","evidence":"Immunopurification and reconstituted ubiquitination from Xenopus extracts; immunofluorescence and antibody microinjection arrest in HeLa cells","pmids":["7736580","7736578"],"confidence":"High","gaps":["Direct enzymatic contribution of CDC16 versus other subunits not resolved","Structural basis of CDC16 incorporation into APC/C unknown"]},{"year":1996,"claim":"Demonstration that cdc16 mutants undergo genome-wide re-replication from normal origins revealed that CDC16/APC/C enforces the once-per-cell-cycle rule for DNA replication, independently of CDK-mediated inhibition.","evidence":"Flow cytometry showing up to 8C DNA content in S. cerevisiae cdc16 mutants; 2D gel and density-transfer confirmation of origin re-firing requiring Cdc6 and Mcm5","pmids":["8620535","9660930"],"confidence":"High","gaps":["Identity of the APC/C substrate(s) whose stabilization permits re-replication not determined","Whether this function is conserved in metazoans not tested"]},{"year":1997,"claim":"Discovery that phosphatase PP5 physically interacts with CDC16's C-terminal TPR block and colocalizes at the mitotic spindle suggested a dephosphorylation-based regulatory input to APC/C activity.","evidence":"Yeast two-hybrid, in vitro binding, deletion mapping, and immunofluorescence colocalization","pmids":["9405394"],"confidence":"Medium","gaps":["Functional consequence of PP5–CDC16 interaction on APC/C activity not demonstrated","No in vivo validation of dephosphorylation"]},{"year":2003,"claim":"Phosphoproteomics revealed CDC16 as a major target of Cdk1-dependent mitotic phosphorylation, and showed that Cdk1 (not Plk1) phosphorylation of APC/C TPR subunits is sufficient for CDC20 binding and APC/C activation.","evidence":"Mass spectrometry phospho-site mapping, in vitro kinase assays with Cdk1 and Plk1, co-immunoprecipitation for CDC20 binding","pmids":["14657031"],"confidence":"High","gaps":["Individual contribution of CDC16 phosphorylation sites versus other TPR subunit sites not resolved","No phospho-site mutagenesis on CDC16 alone"]},{"year":2004,"claim":"Identification of Swm1/Apc13 as a subunit required for stable CDC16 and CDC27 incorporation into the APC/C revealed a hierarchy of accessory subunits that maintain complex integrity and ligase activity.","evidence":"Affinity purification–mass spectrometry, in vitro ubiquitin ligase assay, in vivo cell cycle delay upon SWM1 deletion in S. cerevisiae","pmids":["15060174"],"confidence":"High","gaps":["Structural basis of Swm1-mediated CDC16 incorporation unknown at this time"]},{"year":2009,"claim":"Crystal structures of CDC16/CDC26 complexes resolved the molecular architecture: CDC16 is a 14-TPR superhelix that homo-dimerizes via its N-terminal block, and CDC26 stabilizes APC/C integrity by forming an intermolecular TPR mimic with CDC16's C-terminal domain.","evidence":"X-ray crystallography of S. pombe Cut9–Hcn1 complex, biophysical binding assays, genetic complementation","pmids":["19668213","20924356"],"confidence":"High","gaps":["Full atomic model of CDC16 within the intact human APC/C not yet available at this stage","Mechanism by which N-terminal dimerization contributes to APC/C function not tested in vivo"]},{"year":2022,"claim":"Evidence that non-APC/C proteins (DEPDC1B, c-Jun) can competitively sequester CDC16, disrupting APC/C complex formation, suggested CDC16 availability may be a regulatory node exploited in cancer contexts.","evidence":"Co-immunoprecipitation and ubiquitination assays in melanoma (DEPDC1B–CDC16) and neuroblastoma (c-Jun–CDC16) cell lines, in vivo tumor models","pmids":["35088579","40149013"],"confidence":"Medium","gaps":["Stoichiometry of sequestration relative to total cellular CDC16 pool unclear","Physiological relevance of competitive CDC16 binding outside overexpression settings not established","Independent replication needed"]},{"year":null,"claim":"The individual functional contribution of each CDC16 Cdk1-phosphorylation site, the structural basis of CDC16 within the complete human APC/C holoenzyme at high resolution, and whether CDC16 sequestration by non-APC/C partners represents a physiologically significant regulatory mechanism remain open questions.","evidence":"","pmids":[],"confidence":"High","gaps":["No single-site phosphomutant analysis of CDC16","No cryo-EM structure of human APC/C focused on CDC16-specific contacts at sub-3Å resolution","Physiological relevance of DEPDC1B/c-Jun-mediated CDC16 sequestration not validated in non-cancer settings"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,7]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[9,10]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1,8]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,4]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,2,3,5,8]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,7,11]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[3,5]}],"complexes":["APC/C"],"partners":["CDC27","CDC26","APC13","PP5","DEPDC1B","CDC20"],"other_free_text":[]},"mechanistic_narrative":"CDC16 (APC6) is a TPR-repeat scaffold subunit of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase essential for mitotic progression, anaphase onset, and prevention of DNA re-replication [PMID:7736580, PMID:8620535]. CDC16 forms a contiguous 14-TPR superhelix that homo-dimerizes via its N-terminal TPR block; its C-terminal TPR domain binds CDC26 through an intermolecular TPR mimic that stabilizes overall APC/C integrity [PMID:20924356, PMID:19668213]. Mitosis-specific phosphorylation of CDC16 and other TPR subunits by Cdk1 promotes CDC20 recruitment and APC/C activation, enabling cyclin B ubiquitination and the metaphase-to-anaphase transition [PMID:14657031, PMID:7736578]. CDC16 also restricts DNA replication to once per cell cycle by preventing re-firing of replication origins through a pathway that requires Cdc6 and Mcm5, acting independently of CDK-mediated origin suppression [PMID:9660930]."},"prefetch_data":{"uniprot":{"accession":"Q13042","full_name":"Cell division cycle protein 16 homolog","aliases":["Anaphase-promoting complex subunit 6","APC6","CDC16 homolog","CDC16Hs","Cyclosome subunit 6"],"length_aa":620,"mass_kda":71.7,"function":"Component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle (PubMed:18485873). The APC/C complex acts by mediating ubiquitination and subsequent degradation of target proteins: it mainly mediates the formation of 'Lys-11'-linked polyubiquitin chains and, to a lower extent, the formation of 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains (PubMed:18485873). The APC/C complex catalyzes assembly of branched 'Lys-11'-/'Lys-48'-linked branched ubiquitin chains on target proteins (PubMed:29033132)","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, spindle","url":"https://www.uniprot.org/uniprotkb/Q13042/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CDC16","classification":"Common 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HOMOLOG; MEAK7","url":"https://www.omim.org/entry/619331"},{"mim_id":"618039","title":"TBC1 DOMAIN FAMILY, MEMBER 9B; TBC1D9B","url":"https://www.omim.org/entry/618039"},{"mim_id":"618035","title":"TBC1 DOMAIN FAMILY, MEMBER 9; TBC1D9","url":"https://www.omim.org/entry/618035"},{"mim_id":"616659","title":"TBC1 DOMAIN FAMILY, MEMBER 17; TBC1D17","url":"https://www.omim.org/entry/616659"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CDC16"},"hgnc":{"alias_symbol":["APC6","ANAPC6","CUT9"],"prev_symbol":[]},"alphafold":{"accession":"Q13042","domains":[{"cath_id":"1.25.40.10","chopping":"2-101_122-210","consensus_level":"medium","plddt":88.6665,"start":2,"end":210}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13042","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13042-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13042-F1-predicted_aligned_error_v6.png","plddt_mean":78.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CDC16","jax_strain_url":"https://www.jax.org/strain/search?query=CDC16"},"sequence":{"accession":"Q13042","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13042.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13042/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13042"}},"corpus_meta":[{"pmid":"7736580","id":"PMC_7736580","title":"A 20S complex 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Immunopurified CDC27 complexes (containing CDC16) were sufficient to complement interphase extracts or a mixture of recombinant UBC4 and E1.\",\n      \"method\": \"Biochemical fractionation of Xenopus mitotic egg extracts, immunodepletion, immunopurification, in vitro ubiquitination reconstitution assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of E3 ubiquitin ligase activity, complementation assay, replicated across labs\",\n      \"pmids\": [\"7736580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"S. pombe cdc16 (ortholog of human CDC16/APC6) is essential for cell viability and is required both for maintenance of p34cdc2 kinase activity and for restricting septum formation to once per cell cycle; loss of cdc16 leads to multiple septa without cytokinesis, placing it as a negative regulator of septation.\",\n      \"method\": \"Gene deletion, temperature-sensitive allele analysis, genetic interaction studies in S. pombe\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO/loss-of-function with defined cellular phenotype in orthologous organism, replicated findings\",\n      \"pmids\": [\"8334988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"S. pombe Cdc16, together with Byr4, forms a two-component GTPase-activating protein (GAP) for the Spg1 GTPase that controls septation. Byr4 alone inhibits GTP dissociation from Spg1, but Byr4 and Cdc16 together display Spg1 GAP activity in vitro; Cdc16 alone has no detectable GAP activity. This complex negatively regulates septation by modulating the nucleotide state of Spg1.\",\n      \"method\": \"In vitro GTPase activity assay, yeast two-hybrid, co-precipitation in vitro and in yeast, genetic epistasis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical reconstitution of GAP activity with mutagenesis, complemented by genetic data\",\n      \"pmids\": [\"9742395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Byr4 contains one Cdc16-binding site and four Spg1-binding sites (SBS1-SBS4). The Cdc16-binding site together with SBS4 of Byr4 is necessary and sufficient to reconstitute Cdc16-dependent Spg1 GAP activity. Multiple Spg1-binding sites cooperate to achieve high-affinity Spg1 binding by Byr4.\",\n      \"method\": \"In vitro binding assay, in vitro GTPase assay with Byr4 deletion/point mutants, kinetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with domain-mapping mutagenesis\",\n      \"pmids\": [\"10196225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The S. pombe cut9+ gene (ortholog of CDC16/APC6) encodes a 78 kDa TPR-repeat protein required for the onset of anaphase; cut9 mutants enter mitosis, form short spindles, but block anaphase while continuing postanaphase events (spindle degradation, septation, cytokinesis). Mutation sites reside in the TPR domain. Genetic interactions with nuc2+, scn1, and scn2 place cut9 in the anaphase initiation pathway.\",\n      \"method\": \"Temperature-sensitive mutant analysis, genetic epistasis, gene cloning and sequence analysis in S. pombe\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — defined cellular phenotype with genetic epistasis in orthologous organism, foundational paper\",\n      \"pmids\": [\"7798319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"In S. cerevisiae, CDC16 (along with CDC27) is required to restrict DNA replication to once per cell cycle; cdc16 mutants accumulate up to 8C DNA through overreplication of all chromosomes within a single cell cycle, even while Clb2-Cdc28 kinase activity remains elevated, implicating CDC16 in proteolytic control of an S-phase initiator.\",\n      \"method\": \"Conditional mutant analysis, flow cytometry, fluorescence microscopy in S. cerevisiae\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic loss-of-function with defined replication phenotype, single lab\",\n      \"pmids\": [\"8620535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"CDC16 is required to prevent inappropriate firing of replication origins; cdc16 mutants reinitiate DNA synthesis from normal chromosomal replication origins, and this rereplication requires both Cdc6p and Mcm5p (initiation proteins thought to be inactivated by elevated Cdc28p kinase).\",\n      \"method\": \"2D DNA electrophoresis, density-transfer experiments, genetic analysis in S. cerevisiae\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct demonstration of origin refiring by 2D gel electrophoresis with mechanistic epistasis\",\n      \"pmids\": [\"9660930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The serine/threonine phosphatase PP5 physically interacts with CDC16 and CDC27 (two TPR-containing APC subunits) via its N-terminal TPR domain; the binding site on CDC16 maps to its C-terminal TPR block. PP5 co-localizes with the mitotic spindle apparatus, suggesting it may regulate APC activity through dephosphorylation.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding, deletion mapping, indirect immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — two-hybrid and in vitro binding with localization data, single lab\",\n      \"pmids\": [\"9405394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Swm1/Apc13 is required for the stable association of CDC16 and CDC27 with the APC/C; loss of Swm1/Apc13 destabilizes CDC16 and CDC27 within the complex and abolishes APC/C ubiquitin ligase activity in vitro and delays APC/C-dependent cell cycle events in vivo.\",\n      \"method\": \"Mass spectrometry, in vitro ubiquitination assay, co-immunoprecipitation, genetic complementation across species\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (MS, in vitro ligase assay, Co-IP, in vivo cell cycle analysis)\",\n      \"pmids\": [\"15060174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of S. pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26) reveals that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units: a C-terminal TPR block interacts with Hcn1, and an N-terminal TPR block mediates Cdc16/Cut9 homo-dimerization through a conserved mechanism also used by Cdc27. The acetylated N-terminal Met of Hcn1 is enclosed within the Cut9 TPR chamber, protecting Hcn1 from Doa10-mediated ubiquitin-dependent degradation.\",\n      \"method\": \"X-ray crystallography, structural analysis, functional interpretation of domain organization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mechanistic interpretation validated by domain architecture\",\n      \"pmids\": [\"20924356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CDC26 stabilizes the structure of APC6 (CDC16) through an intermolecular TPR mimic composed of one helix from each protein; biophysical, structural, and genetic studies show this interaction is essential for APC assembly and integrity.\",\n      \"method\": \"X-ray crystallography, biophysical assays, genetic analysis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biophysical and genetic validation in a single study\",\n      \"pmids\": [\"19668213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DEPDC1B sequesters human CDC16 (an APC/C subunit and E3 ubiquitin ligase component) away from its substrate SCUBE3, preventing SCUBE3 ubiquitination and proteasomal degradation, thereby promoting secretion of SCUBE3 and melanoma angiogenesis and metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression in melanoma cells and in vivo orthotopic models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with functional ubiquitination assay, single lab, novel substrate interaction\",\n      \"pmids\": [\"35088579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"c-Jun competitively interacts with CDC16, a key APC subunit, thereby reducing APC/C complex formation and inhibiting cell cycle progression, which promotes neuroblastoma cell differentiation.\",\n      \"method\": \"Co-immunoprecipitation, cell cycle analysis by flow cytometry, EdU proliferation assay, transwell migration assay\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with phenotypic readouts, single lab, minimal mechanistic follow-up\",\n      \"pmids\": [\"40149013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"YDJC interacts with CDC16 via co-immunoprecipitation; CDC16 overexpression suppresses EMT and induces PP2A expression, while CDC16 promotes ubiquitination of YDJC. When YDJC is overexpressed it escapes CDC16 interaction and promotes ubiquitination of PP2A.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, ubiquitination assay in lung cancer cells\",\n      \"journal\": \"Journal of oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP-based interaction study with limited mechanistic dissection, single lab\",\n      \"pmids\": [\"31485224\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Human CDC16 (APC6) is a TPR-superhelix subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase that is essential for mitotic progression: it homo-dimerizes through its N-terminal TPR block, binds CDC26 through an intermolecular TPR mimic that stabilizes its structure and APC assembly, is held within the APC by Swm1/Apc13, and together with other APC subunits catalyzes ubiquitin conjugation to substrates such as cyclin B to drive anaphase onset and prevent DNA rereplication; in S. pombe its ortholog additionally forms a two-component Spg1 GAP with Byr4 to negatively regulate cytokinesis.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper and extract discoveries only from KEEP papers.\n\n**Classification notes:**\n- Papers 1, 3 (retrieved) and 27 (curated) = KEEP: directly about CDC16/APC6 as APC/C subunit\n- Paper 4 (retrieved) = EXCLUDE (alias collision): S. pombe Cdc16 is a GAP for Spg1 — this is a *different* S. pombe gene (the septation regulator, not the APC subunit ortholog). Wait — re-examining: the S. pombe APC subunit is Cut9/Cdc16, while the septation regulator is also called Cdc16 in S. pombe. The corpus shows paper 6 (cut9/cdc16 = APC subunit ortholog) and paper 3 (S. pombe cdc16 = septation regulator with GAP activity). These are clearly two different S. pombe genes with the same name. The APC subunit CDC16 is the human gene in question. Papers 4, 21, 23 describe the S. pombe septation regulator (GAP for Spg1) — EXCLUDE as alias collision.\n- Paper 6 (cut9+) = KEEP: cut9/Cdc16 is the S. pombe APC subunit ortholog\n- Paper 12 = KEEP: crystal structure of Cut9/Cdc16 (APC subunit)\n- Paper 16 = KEEP: CDC26-APC6 structure\n- Paper 9 = KEEP: PP5 interacts with CDC16/CDC27\n- Paper 14 = KEEP: Swm1/Apc13 stabilizes Cdc16/Cdc27 association\n- Paper 8 = KEEP: CDC16 in DNA replication control\n- Paper 20 = KEEP: CDC16 controls replication origins\n- Paper 7 = KEEP (plant APC6/CDC16 ortholog — consistent function)\n- Paper 22 = KEEP: DEPDC1B-CDC16-SCUBE3 (human CDC16 as APC/C ubiquitin ligase)\n- Paper 27-29 (retrieved) = EXCLUDE: TBC domain papers, alias collisions\n- Paper 5, 10, 11, 13, 15, 18, 19 = EXCLUDE: TBC domain alias collisions\n- Paper 29 (retrieved) = KEEP: c-Jun-CDC16 interaction\n- Paper 28 (retrieved) = KEEP: YDJC-CDC16 (2019)\n- Paper 30 (preprint) = EXCLUDE: Arabidopsis\n- Paper 31, 32 (preprints) = EXCLUDE: TBC domain alias collisions\n- Curated papers: most are general APC/C or interactome papers; include those with direct CDC16 mechanistic findings\n- Curated 25 = KEEP: APC phosphorylation including CDC16\n- Curated 27 = KEEP: CDC16Hs localization\n- Curated 11 = KEEP: APC/C inhibition (CDC16 as subunit context)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"CDC16 (human homolog of S. cerevisiae Cdc16) is a subunit of the 20S anaphase-promoting complex (APC), a mitosis-specific ubiquitin-protein ligase that catalyzes conjugation of ubiquitin to cyclin B, targeting it for proteasomal degradation at the onset of anaphase. Immunopurified CDC27-containing complexes (which include CDC16) are sufficient to reconstitute cyclin B ubiquitination activity.\",\n      \"method\": \"Biochemical fractionation of mitotic Xenopus egg extracts, immunodepletion with CDC27 antibodies, immunopurification, in vitro ubiquitination reconstitution assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro ubiquitin ligase activity, foundational study replicated widely\",\n      \"pmids\": [\"7736580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Human CDC16Hs (encoded by a cDNA homologous to S. cerevisiae CDC16) colocalizes with CDC27Hs to the centrosome throughout the mammalian cell cycle and to the mitotic spindle. Microinjection of anti-CDC27Hs antibodies arrests HeLa cells in metaphase, demonstrating that the CDC16/CDC27-containing complex is essential for the metaphase-to-anaphase transition.\",\n      \"method\": \"Immunofluorescence microscopy, antibody microinjection into HeLa cells, cell cycle arrest assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with functional (arrest) consequence, replicated in complementary study\",\n      \"pmids\": [\"7736578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The fission yeast cut9+ gene (encoding the S. pombe ortholog of Cdc16/APC6) is required for the initiation of anaphase; temperature-sensitive cut9 mutants enter mitosis and form short spindles but block at anaphase onset, while postanaphase events (spindle disassembly, septation, cytokinesis) continue inappropriately. Cut9 is a 78-kDa TPR-containing protein essential for viability.\",\n      \"method\": \"Temperature-sensitive mutant analysis, genetic epistasis (suppressor genes nuc2+, scn1, scn2), microscopy of cell cycle progression\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis and phenotypic analysis in a clear ortholog, strong evidence\",\n      \"pmids\": [\"7798319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CDC16 and CDC27 in S. cerevisiae are required to restrict DNA replication to once per cell cycle; conditional cdc16 mutants rereplicate all chromosomes (up to 8C DNA content) within a single cell cycle without passing through mitosis, despite persistently elevated Clb2-Cdc28 (CDK) kinase activity, indicating CDC16 prevents rereplication independently of CDK-mediated inhibition of re-initiation.\",\n      \"method\": \"Conditional mutant analysis (temperature-sensitive alleles), flow cytometry (DNA content), genetic screen\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean loss-of-function with defined molecular phenotype, mechanistic follow-up\",\n      \"pmids\": [\"8620535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The serine/threonine phosphatase PP5 physically interacts with CDC16 and CDC27, two TPR-containing subunits of the APC/C. The interaction requires only the N-terminal TPR-containing domain of PP5, while the binding site on CDC16 and CDC27 maps to their C-terminal TPR block. PP5 colocalizes with CDC16/CDC27 at the mitotic spindle apparatus, suggesting it may regulate APC/C activity through dephosphorylation.\",\n      \"method\": \"Yeast two-hybrid analysis, in vitro binding assay, deletion mapping, indirect immunofluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — two-hybrid plus in vitro binding with localization data, single lab\",\n      \"pmids\": [\"9405394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"In S. cerevisiae cdc16 mutants, rereplication initiates specifically from normal chromosomal replication origins, and requires both the initiation proteins Cdc6p and Cdc46/Mcm5p — proteins thought to be inactivated by CDK. This establishes that CDC16 is required to prevent inappropriate firing of replication origins after S-phase, acting independently of CDK-mediated origin suppression.\",\n      \"method\": \"Two-dimensional DNA gel electrophoresis, density-transfer experiments, genetic epistasis (cdc6 and cdc46 double mutants)\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (2D gels, density transfer, epistasis) establishing mechanistic pathway\",\n      \"pmids\": [\"9660930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The Arabidopsis NOMEGA gene encodes the plant ortholog of APC6/CDC16; nomega mutant embryo sacs arrest at the two-nucleate stage and are unable to degrade Cyclin B, directly demonstrating that the CDC16-containing APC/C is required for Cyclin B proteolysis during cell cycle progression in plant gametophyte development.\",\n      \"method\": \"Genetic mutant characterization, cyclin B degradation assay in vivo, complementation\",\n      \"journal\": \"The Plant journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional assay (cyclin B stability) in loss-of-function mutant, ortholog context\",\n      \"pmids\": [\"14675450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Swm1/Apc13 is an evolutionarily conserved APC/C subunit that promotes stable association of CDC16 and CDC27 with the complex; deletion of SWM1 reduces the incorporation of Cdc16 and Cdc27 into the APC/C, abolishes ubiquitin ligase activity in vitro, and delays APC/C-dependent cell cycle events in vivo.\",\n      \"method\": \"Affinity purification, mass spectrometry, in vitro ubiquitin ligase activity assay, in vivo cell cycle analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (AP-MS, in vitro ligase assay, in vivo phenotype) establishing subunit interaction requirement\",\n      \"pmids\": [\"15060174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"At least 34 mitosis-specific phosphorylation sites exist on human APC/C subunits; 32 of these cluster in Apc1 and TPR subunits including CDC16 (Cdc16), CDC27, CDC23, and Apc7. Cdk1 can generate at least 15 mitotic phospho-sites in vitro. APC phosphorylation by Cdk1 (but not Plk1) is sufficient for increased CDC20 binding and APC activation. Phospho-APC accumulates at centrosomes in prometaphase where cyclin B ubiquitination initiates.\",\n      \"method\": \"Mass spectrometry phospho-site mapping, in vitro kinase assays (Cdk1, Plk1), co-immunoprecipitation, immunofluorescence with phospho-specific antibodies\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — phospho-site mapping combined with in vitro kinase assays and localization, multiple methods\",\n      \"pmids\": [\"14657031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The crystal structure of S. pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26) reveals that Cdc16/Cut9 is a contiguous superhelix of 14 TPR units. The C-terminal TPR block interacts with Hcn1/Cdc26, while the N-terminal TPR block mediates CDC16 self-association through a homotypic dimer interface structurally related to the Cdc27 dimerization domain. The acetylated N-terminal Met of Hcn1 is enclosed within the Cut9 TPR chamber, protecting Hcn1 from Doa10-mediated ubiquitin-dependent degradation.\",\n      \"method\": \"X-ray crystallography, biochemical characterization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with functional interpretation and mechanistic validation\",\n      \"pmids\": [\"20924356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Biophysical and structural studies show that CDC26 stabilizes the structure of APC6 (CDC16) through an intermolecular TPR mimic composed of one helix from each protein; genetic studies confirm that this interaction is required for APC integrity.\",\n      \"method\": \"X-ray crystallography, biophysical binding assays, genetic complementation\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with genetic validation, mechanistic explanation for CDC26 function\",\n      \"pmids\": [\"19668213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DEPDC1B competitively associates with human CDC16 (an APC/C ubiquitin ligase subunit) to sequester it, thereby preventing CDC16-mediated ubiquitination and proteasomal degradation of the secreted protein SCUBE3. This stabilization of SCUBE3 promotes melanoma angiogenesis and metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression rescue, in vivo tumor/angiogenesis models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP plus ubiquitination assay and in vivo models, single lab\",\n      \"pmids\": [\"35088579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"YDJC physically interacts with CDC16 as shown by co-immunoprecipitation; CDC16 siRNA knockdown induces sphingosylphosphorylcholine-triggered keratin reorganization, migration, and invasion in A549 lung cancer cells, while CDC16 overexpression blocks these events, suggesting CDC16 suppresses EMT-related signaling.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, migration/invasion assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP plus knockdown/overexpression phenotype, no direct mechanistic pathway placement\",\n      \"pmids\": [\"29796162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CDC16 overexpression induces ubiquitination of YDJC protein and increases PP2A expression; YDJC overexpression escapes CDC16-mediated ubiquitination (dependent on YDJC deacetylase activity) and promotes PP2A ubiquitination, leading to EMT in lung cancer cells. This places CDC16 as a ubiquitin ligase regulator in a YDJC-PP2A-ERK2-EMT axis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression with deacetylase-dead mutant (YDJCD13A), in vivo orthotopic mouse model\",\n      \"journal\": \"Journal of oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — mechanistic claims based on overexpression and co-IP, single lab, limited controls\",\n      \"pmids\": [\"31485224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"c-Jun competitively interacts with CDC16, a key APC/C subunit, reducing APC complex formation and inhibiting cell cycle progression from G1 in neuroblastoma cells. c-Jun overexpression inhibits neuroblastoma cell proliferation and migration via sequestration of CDC16.\",\n      \"method\": \"Co-immunoprecipitation, flow cytometry (cell cycle), EdU proliferation assay, transwell migration assay, retinoic acid treatment\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP with cellular phenotype assays, single lab, limited mechanistic depth\",\n      \"pmids\": [\"40149013\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CDC16/APC6 is a TPR-repeat scaffold subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase: it forms a 14-TPR superhelix that homo-dimerizes via its N-terminal TPR block, binds CDC26 via an intermolecular TPR mimic to stabilize APC/C integrity, and is phosphorylated by Cdk1 at mitosis-specific sites to enable APC/C activation and CDC20 recruitment, collectively driving ubiquitination of cyclin B and other substrates to trigger anaphase onset and prevent DNA re-replication.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CDC16 (APC6/Cut9) is a TPR-repeat subunit of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase essential for mitotic progression, anaphase onset, and prevention of DNA rereplication. As part of the APC/C, CDC16 contributes to the ubiquitin-dependent destruction of cell cycle regulators including cyclin B; loss of CDC16 function causes anaphase arrest and inappropriate refiring of replication origins [PMID:7736580, PMID:7798319, PMID:8620535, PMID:9660930]. Structurally, CDC16 forms a contiguous 14-TPR superhelix that homo-dimerizes through its N-terminal TPR block and binds CDC26 through an intermolecular TPR mimic that stabilizes CDC16 folding and APC/C assembly, while the accessory subunit Swm1/Apc13 is required for stable CDC16 incorporation into the complex [PMID:20924356, PMID:19668213, PMID:15060174]. In S. pombe, the CDC16 ortholog additionally partners with Byr4 to form a two-component GAP for the Spg1 GTPase, negatively regulating septation and cytokinesis [PMID:9742395, PMID:10196225].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Establishing that cdc16 is essential for viability and negatively regulates septation answered the question of whether cdc16 functions beyond generic cell division control, revealing a specific role in restricting septum formation to once per cycle.\",\n      \"evidence\": \"Temperature-sensitive and deletion alleles in S. pombe showing multiple-septation phenotype\",\n      \"pmids\": [\"8334988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of cdc16 in septation was unknown\", \"No biochemical activity assigned\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Identification of Cut9 (CDC16 ortholog) as a TPR-repeat protein required specifically for anaphase onset established the structural family of the protein and placed it in the chromosome-segregation pathway.\",\n      \"evidence\": \"Temperature-sensitive cut9 mutants in S. pombe blocking anaphase; sequence analysis revealing TPR repeats\",\n      \"pmids\": [\"7798319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No direct enzymatic activity was demonstrated\", \"Whether Cut9 was part of a larger complex was unknown\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Demonstrating that CDC16 is a core subunit of the 20S APC that functions as a mitosis-specific E3 ubiquitin ligase for cyclin B destruction unified its anaphase-promoting role with the ubiquitin-proteasome pathway.\",\n      \"evidence\": \"Biochemical fractionation and immunopurification from Xenopus egg extracts; in vitro ubiquitination reconstitution\",\n      \"pmids\": [\"7736580\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct catalytic contribution of CDC16 versus other APC subunits was unresolved\", \"Substrate scope beyond cyclin B unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showing that cdc16 mutants undergo DNA rereplication up to 8C revealed a second essential APC/C function: preventing re-licensing of replication origins within a single cell cycle.\",\n      \"evidence\": \"Flow cytometry and microscopy of conditional cdc16 mutants in S. cerevisiae\",\n      \"pmids\": [\"8620535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the proteolytic substrate whose persistence caused rereplication was not established\", \"Whether this was a direct CDC16 function or general APC/C function was unclear\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Two advances resolved distinct CDC16 activities: (1) 2D gel electrophoresis showed cdc16-dependent rereplication fires from normal origins requiring Cdc6p/Mcm5p, linking APC/C to origin licensing control; (2) in vitro reconstitution showed S. pombe Cdc16 forms a two-component Spg1 GAP with Byr4, explaining its anti-septation function through direct GTPase regulation.\",\n      \"evidence\": \"2D DNA gel electrophoresis and density-transfer in S. cerevisiae; in vitro GTPase assay and yeast two-hybrid/co-precipitation in S. pombe\",\n      \"pmids\": [\"9660930\", \"9742395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the GAP function is conserved beyond fission yeast was unknown\", \"Structural basis of the Cdc16-Byr4 interaction was unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping Byr4's Cdc16-binding site and showing it cooperates with a single Spg1-binding site to reconstitute GAP activity defined the minimal functional unit of the Cdc16-Byr4 GAP.\",\n      \"evidence\": \"In vitro GTPase assay with Byr4 deletion/point mutants and kinetic analysis\",\n      \"pmids\": [\"10196225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of the ternary Cdc16-Byr4-Spg1 complex\", \"Regulation of Cdc16-Byr4 assembly during cytokinesis was unexplored\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrating that Swm1/Apc13 is required for stable CDC16 association with the APC/C answered how CDC16 is held within the holoenzyme and showed its loss abolishes APC/C ligase activity.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, in vitro ubiquitination, and cross-species complementation\",\n      \"pmids\": [\"15060174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise binding interface between Swm1 and CDC16 was unknown\", \"Whether Swm1 loss phenocopies cdc16 loss fully was not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Crystal structure of CDC26 bound to APC6 revealed that CDC26 stabilizes CDC16 through an intermolecular TPR mimic — one helix from each protein — explaining why CDC26 is essential for APC assembly.\",\n      \"evidence\": \"X-ray crystallography with biophysical and genetic validation\",\n      \"pmids\": [\"19668213\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length CDC16 structure in the context of holo-APC/C was not determined\", \"Dynamics of the CDC16-CDC26 interaction during the cell cycle unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The crystal structure of S. pombe Cut9 (full-length CDC16) revealed a 14-TPR superhelix architecture, showed homo-dimerization through the N-terminal TPR block, and explained how the CDC26 N-terminal methionine is enclosed in a TPR chamber to protect it from degradation.\",\n      \"evidence\": \"X-ray crystallography of Cut9-Hcn1 complex from S. pombe\",\n      \"pmids\": [\"20924356\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CDC16 dimerization contributes to APC/C architecture in the context of cryo-EM structures was not yet integrated\", \"Post-translational regulation of CDC16 TPR domains was unstudied\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of SCUBE3 as a CDC16-dependent ubiquitination substrate in melanoma, sequestered away from CDC16 by DEPDC1B, extended APC/C substrate recognition to a secreted angiogenic factor.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, siRNA knockdown, overexpression in melanoma cells and in vivo models\",\n      \"pmids\": [\"35088579\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SCUBE3 is a direct APC/C substrate or requires an adaptor is unknown\", \"Not independently replicated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CDC16 contributes to substrate selectivity within the APC/C holoenzyme, the full repertoire of CDC16-dependent APC/C substrates in human cells, and whether CDC16 has APC/C-independent functions in vertebrates remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No systematic identification of CDC16-specific substrates in human cells\", \"Role of CDC16 phosphorylation in APC/C activation is incompletely characterized\", \"Vertebrate CDC16 functions outside the APC/C context have not been demonstrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 8, 11]},\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 8, 11]}\n    ],\n    \"complexes\": [\n      \"APC/C\",\n      \"Cdc16-Byr4 GAP complex\"\n    ],\n    \"partners\": [\n      \"CDC27\",\n      \"CDC26\",\n      \"SWM1\",\n      \"BYR4\",\n      \"SPG1\",\n      \"PP5\",\n      \"DEPDC1B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CDC16 (APC6) is a TPR-repeat scaffold subunit of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase essential for mitotic progression, anaphase onset, and prevention of DNA re-replication [PMID:7736580, PMID:8620535]. CDC16 forms a contiguous 14-TPR superhelix that homo-dimerizes via its N-terminal TPR block; its C-terminal TPR domain binds CDC26 through an intermolecular TPR mimic that stabilizes overall APC/C integrity [PMID:20924356, PMID:19668213]. Mitosis-specific phosphorylation of CDC16 and other TPR subunits by Cdk1 promotes CDC20 recruitment and APC/C activation, enabling cyclin B ubiquitination and the metaphase-to-anaphase transition [PMID:14657031, PMID:7736578]. CDC16 also restricts DNA replication to once per cell cycle by preventing re-firing of replication origins through a pathway that requires Cdc6 and Mcm5, acting independently of CDK-mediated origin suppression [PMID:9660930].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Identification of the fission yeast ortholog Cut9 as a TPR protein essential for anaphase initiation established that CDC16-family genes are required for the metaphase-to-anaphase transition.\",\n      \"evidence\": \"Temperature-sensitive cut9 mutants in S. pombe arrest at anaphase onset; genetic epistasis with nuc2+, scn1, scn2\",\n      \"pmids\": [\"7798319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical function of Cut9/CDC16 unknown\", \"No direct connection to ubiquitin-dependent proteolysis at this stage\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Biochemical purification of the APC/C as a cyclin B ubiquitin ligase containing CDC16 and localization of CDC16 to the centrosome and mitotic spindle established CDC16 as a core subunit of the mitosis-specific E3 ligase driving anaphase.\",\n      \"evidence\": \"Immunopurification and reconstituted ubiquitination from Xenopus extracts; immunofluorescence and antibody microinjection arrest in HeLa cells\",\n      \"pmids\": [\"7736580\", \"7736578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic contribution of CDC16 versus other subunits not resolved\", \"Structural basis of CDC16 incorporation into APC/C unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstration that cdc16 mutants undergo genome-wide re-replication from normal origins revealed that CDC16/APC/C enforces the once-per-cell-cycle rule for DNA replication, independently of CDK-mediated inhibition.\",\n      \"evidence\": \"Flow cytometry showing up to 8C DNA content in S. cerevisiae cdc16 mutants; 2D gel and density-transfer confirmation of origin re-firing requiring Cdc6 and Mcm5\",\n      \"pmids\": [\"8620535\", \"9660930\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the APC/C substrate(s) whose stabilization permits re-replication not determined\", \"Whether this function is conserved in metazoans not tested\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Discovery that phosphatase PP5 physically interacts with CDC16's C-terminal TPR block and colocalizes at the mitotic spindle suggested a dephosphorylation-based regulatory input to APC/C activity.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, deletion mapping, and immunofluorescence colocalization\",\n      \"pmids\": [\"9405394\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of PP5–CDC16 interaction on APC/C activity not demonstrated\", \"No in vivo validation of dephosphorylation\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Phosphoproteomics revealed CDC16 as a major target of Cdk1-dependent mitotic phosphorylation, and showed that Cdk1 (not Plk1) phosphorylation of APC/C TPR subunits is sufficient for CDC20 binding and APC/C activation.\",\n      \"evidence\": \"Mass spectrometry phospho-site mapping, in vitro kinase assays with Cdk1 and Plk1, co-immunoprecipitation for CDC20 binding\",\n      \"pmids\": [\"14657031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Individual contribution of CDC16 phosphorylation sites versus other TPR subunit sites not resolved\", \"No phospho-site mutagenesis on CDC16 alone\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of Swm1/Apc13 as a subunit required for stable CDC16 and CDC27 incorporation into the APC/C revealed a hierarchy of accessory subunits that maintain complex integrity and ligase activity.\",\n      \"evidence\": \"Affinity purification–mass spectrometry, in vitro ubiquitin ligase assay, in vivo cell cycle delay upon SWM1 deletion in S. cerevisiae\",\n      \"pmids\": [\"15060174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Swm1-mediated CDC16 incorporation unknown at this time\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Crystal structures of CDC16/CDC26 complexes resolved the molecular architecture: CDC16 is a 14-TPR superhelix that homo-dimerizes via its N-terminal block, and CDC26 stabilizes APC/C integrity by forming an intermolecular TPR mimic with CDC16's C-terminal domain.\",\n      \"evidence\": \"X-ray crystallography of S. pombe Cut9–Hcn1 complex, biophysical binding assays, genetic complementation\",\n      \"pmids\": [\"19668213\", \"20924356\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full atomic model of CDC16 within the intact human APC/C not yet available at this stage\", \"Mechanism by which N-terminal dimerization contributes to APC/C function not tested in vivo\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Evidence that non-APC/C proteins (DEPDC1B, c-Jun) can competitively sequester CDC16, disrupting APC/C complex formation, suggested CDC16 availability may be a regulatory node exploited in cancer contexts.\",\n      \"evidence\": \"Co-immunoprecipitation and ubiquitination assays in melanoma (DEPDC1B–CDC16) and neuroblastoma (c-Jun–CDC16) cell lines, in vivo tumor models\",\n      \"pmids\": [\"35088579\", \"40149013\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry of sequestration relative to total cellular CDC16 pool unclear\", \"Physiological relevance of competitive CDC16 binding outside overexpression settings not established\", \"Independent replication needed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The individual functional contribution of each CDC16 Cdk1-phosphorylation site, the structural basis of CDC16 within the complete human APC/C holoenzyme at high resolution, and whether CDC16 sequestration by non-APC/C partners represents a physiologically significant regulatory mechanism remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No single-site phosphomutant analysis of CDC16\", \"No cryo-EM structure of human APC/C focused on CDC16-specific contacts at sub-3Å resolution\", \"Physiological relevance of DEPDC1B/c-Jun-mediated CDC16 sequestration not validated in non-cancer settings\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [9, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 7, 11]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"complexes\": [\n      \"APC/C\"\n    ],\n    \"partners\": [\n      \"CDC27\",\n      \"CDC26\",\n      \"APC13\",\n      \"PP5\",\n      \"DEPDC1B\",\n      \"CDC20\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}