{"gene":"CDC23","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1990,"finding":"CDC23 encodes a protein containing a novel repeating 34-amino acid motif (the TPR motif) reiterated several times; this motif defines a new family of proteins shared among genes required for mitosis and RNA synthesis, and is predicted to consist of amphipathic alpha-helical regions punctuated by proline-induced turns.","method":"Quantitative sequence analysis, cloning and characterization of CDC23 gene","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — foundational structural discovery, highly cited, replicated across multiple proteins","pmids":["2404612"],"is_preprint":false},{"year":1990,"finding":"The CDC23 gene of S. cerevisiae encodes a protein of 626 amino acids with four contiguous TPR repeats and a potential Ca2+-binding site near its N-terminus, expressed as a 2.4–2.5 kb transcript.","method":"Cloning, nucleotide sequencing, and ORF analysis","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 — direct sequence characterization, single study","pmids":["2205535"],"is_preprint":false},{"year":1996,"finding":"Yeast CDC23 directly interacts with the nuclear protein SIN1 (a transcriptional repressor involved in mitotic chromosome segregation); the N-terminus of SIN1 is sufficient to bind the TPR repeat domains of CDC23 in vitro.","method":"Yeast two-hybrid system, in vitro binding with recombinant proteins","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 3 — two-hybrid plus in vitro binding, single lab","pmids":["8710860"],"is_preprint":false},{"year":1998,"finding":"Human CDC23 encodes a 591-amino acid, 68.3 kDa protein with 9 TPR units, mapping to chromosome 5q31; it is a component of the anaphase-promoting complex (APC/C), ubiquitously expressed, with 30% identity and 51% similarity to S. cerevisiae Cdc23.","method":"cDNA cloning, genomic mapping, sequence analysis, Northern blot","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 — molecular cloning and characterization, single study","pmids":["9790767"],"is_preprint":false},{"year":1998,"finding":"The APC/C subunits Cdc23 and Cdc27 are required for the degradation of the APC activator Cdc20 during S phase and early mitosis; this degradation occurs through a mechanism that does not depend on the Cdc20 destruction box, revealing a D-box-independent proteolytic pathway mediated by these APC components.","method":"Genetic analysis in S. cerevisiae, cell cycle protein stability assays, mutant analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis combined with biochemical stability assays, moderately cited","pmids":["9651679"],"is_preprint":false},{"year":1999,"finding":"Fission yeast Cut23 (the S. pombe homolog of Cdc23/APC8) is identified as a subunit of the APC/cyclosome; it contains TPR repeats, its levels fluctuate during the cell cycle (possibly regulated by ubiquitination and degradation), and it is required for metaphase-anaphase progression. Cut23 interacts with polo kinase Plo1 through its TPR domain.","method":"Temperature-sensitive mutant screen, sucrose gradient centrifugation, genetic analysis","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2–3 — biochemical fractionation plus genetic analysis, single study","pmids":["10526233"],"is_preprint":false},{"year":2001,"finding":"The small GTPase Rac forms a complex with the APC component CDC23 in mammalian cell lysates, suggesting that activated Rac may regulate APC/C function or subcellular localization through direct interaction with CDC23.","method":"Co-immunoprecipitation from mammalian cell lysates, yeast two-hybrid","journal":"International journal of molecular medicine","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP, limited mechanistic follow-up","pmids":["11445862"],"is_preprint":false},{"year":2002,"finding":"The APC/C subunit Cdc23 directly interacts with the destruction box (D-box) of the mitotic cyclin Clb2, but not with D-boxes of Clb1, Clb3, or Clb5; mutations in the Clb2 D-box inhibit interaction with Cdc23 both in vivo and in vitro, providing the first evidence for direct APC/C substrate recognition by a specific APC subunit.","method":"Yeast two-hybrid, in vitro binding assay, D-box mutant analysis","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2–3 — two-hybrid plus in vitro binding with mutagenesis, single lab","pmids":["12413490"],"is_preprint":false},{"year":2002,"finding":"Fission yeast polo-like kinase Plo1 physically interacts with the APC/cyclosome through the noncatalytic polo-box domain of Plo1 and the TPR domain of Cut23 (APC8/Cdc23 homolog); a cut23 mutation that specifically disrupts the Plo1 interaction causes metaphase arrest that can be rescued by high Plo1 expression, indicating this interaction is required for mitotic progression.","method":"Co-immunoprecipitation, genetic analysis with cut23 separation-of-function mutation, rescue by Plo1 overexpression","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus genetic rescue with defined phenotype, moderately cited","pmids":["11777938"],"is_preprint":false},{"year":2003,"finding":"Mitotic phosphorylation of the human APC/C occurs on at least 43 sites, with 32 sites clustered in Apc1 and the TPR subunits Cdc27, Cdc16, Cdc23, and Apc7; at least 15 mitosis-specific sites are generated by CDK1 and 3 by Plk1 in vitro. CDK1-mediated APC phosphorylation (but not Plk1) is sufficient for increased Cdc20 binding and APC activation.","method":"Mass spectrometry phosphoproteomics, in vitro kinase assays with CDK1 and Plk1, Cdc20 binding assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay plus mass spectrometry phosphosite mapping, highly cited","pmids":["14657031"],"is_preprint":false},{"year":2008,"finding":"The human APC/C assembles K11-linked ubiquitin chains on substrates; the TEK-box surface of ubiquitin, homologous to motifs in APC/C substrates, enables the APC/C to switch from substrate lysines to ubiquitin lysines during chain elongation. CDC23 (as part of the APC/C) participates in this ubiquitin chain assembly mechanism.","method":"In vitro ubiquitination assays, ubiquitin chain topology analysis, mutagenesis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro ubiquitination with mutagenesis, highly cited","pmids":["18485873"],"is_preprint":false},{"year":2011,"finding":"Functional knockdown of CDC23 in human thyroid cancer cells causes accumulation of cells in S and G2/M phases, inhibits proliferation, tumor spheroid formation, and anchorage-independent growth, with significant accumulation of cyclin B1 and securin protein; triple knockdown of CDC23, cyclin B1, and securin reverses the proliferation and cell cycle effects, placing CDC23 upstream of cyclin B1 and securin degradation.","method":"siRNA knockdown, cell cycle analysis by flow cytometry, western blotting, triple knockdown rescue experiment, in vitro proliferation and colony assays","journal":"Endocrine-related cancer","confidence":"Medium","confidence_rationale":"Tier 2 — KD with defined phenotype and epistasis rescue, single lab","pmids":["21990323"],"is_preprint":false},{"year":2016,"finding":"The crystal structure of the N-terminal domain of human Cdc23 (Cdc23(Nterm)), a subunit of the APC/C, was determined by sulfur SAD phasing to 3.1 Å resolution, providing structural insight into the TPR-containing APC/C subunit.","method":"X-ray crystallography, sulfur SAD phasing","journal":"Acta crystallographica. Section D, Structural biology","confidence":"Medium","confidence_rationale":"Tier 1 — crystal structure determination, limited functional validation in this paper","pmids":["26960127"],"is_preprint":false},{"year":2023,"finding":"Homozygous missense variants in CDC23 (p.Y329C and p.R330C) cause female infertility with oocyte maturation defects; p.Y329C decreases CDC23 protein level, while p.R330C alters CDC23 localization in HeLa cells and mouse oocytes. Knock-in mice (Cdc23Y329C/Y329C) show low CDC23 and APC4 levels and accumulation of securin and cyclin B1 in oocytes; treatment with AZ3146 (a Mps1 kinase inhibitor) rescues the phenotype.","method":"Patient variant identification, in vitro expression assays, immunofluorescence localization, knock-in mouse model, western blotting, pharmacological rescue","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including mouse knock-in, localization, biochemical analysis, and pharmacological rescue","pmids":["37768355"],"is_preprint":false},{"year":2024,"finding":"The C-terminal disordered loop of APC8 (Cdc23 homolog) directly recruits the CDK adaptor protein Xe-p9/Cks2, positioning the CDK-cyclin B complex near the autoinhibitory Apc1-300L loop; this stimulates phosphorylation and removal of Apc1-300L to form active APC/C-Cdc20. Apc8-L acts synergistically with Apc3-L (Cdc27 loop) for full mitotic APC/C activation.","method":"Systematic loop-deletion mutant analysis of APC/C, biochemical reconstitution, protein interaction assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 — reconstituted biochemistry with systematic mutagenesis of loop domains, mechanistic validation","pmids":["38776225"],"is_preprint":false},{"year":2024,"finding":"Comparative cryo-EM structures of S. cerevisiae APC/C reveal that APC8 (Cdc23) contributes to overall scaffold architecture; unlike in human APC/C where the CDC20 C-box binding site of APC8 is sterically blocked by the phospho-regulatable Apc1 autoinhibitory segment, S. cerevisiae APC/C shows no evidence of this autoinhibitory segment blocking APC8, indicating species-specific differences in coactivator-mediated APC/C activation mechanisms.","method":"Cryo-EM structure determination of multiple S. cerevisiae APC/C complexes, comparative structural analysis with human APC/C","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — cryo-EM structures, preprint without peer review","pmids":["bio_10.1101_2024.06.19.599685"],"is_preprint":true}],"current_model":"CDC23 (human APC8) is a TPR-repeat-containing core subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase that undergoes CDK1-mediated mitotic phosphorylation to enable Cdc20 binding and APC/C activation; its C-terminal disordered loop recruits CDK adaptor Cks2 to stimulate phosphorylation of the Apc1 autoinhibitory loop, its TPR domain mediates interaction with polo-like kinase for mitotic progression, it directly contacts the destruction box of substrates such as cyclin Clb2, it controls degradation of Cdc20 and the accumulation of cyclin B1 and securin, and loss-of-function variants cause female infertility through oocyte maturation defects associated with securin and cyclin B1 accumulation."},"narrative":{"teleology":[{"year":1990,"claim":"Identification of the TPR motif in CDC23 established a structural framework for understanding how a repetitive α-helical fold could underpin protein-protein interactions in cell cycle regulators.","evidence":"Quantitative sequence analysis and cloning of yeast CDC23 revealing iterated 34-amino acid repeats","pmids":["2404612","2205535"],"confidence":"High","gaps":["No functional assay linking TPR repeats to APC/C activity","Mammalian homolog not yet cloned"]},{"year":1998,"claim":"Cloning of human CDC23 and its assignment as an APC/C subunit, combined with genetic evidence that Cdc23 controls Cdc20 degradation, placed CDC23 within the ubiquitin-dependent cell cycle machinery.","evidence":"cDNA cloning and Northern blot of human CDC23; genetic stability assays in S. cerevisiae showing Cdc23/Cdc27-dependent, D-box-independent Cdc20 turnover","pmids":["9790767","9651679"],"confidence":"High","gaps":["Mechanism of D-box-independent Cdc20 degradation unknown","Physical interaction between CDC23 and substrates not yet shown"]},{"year":2002,"claim":"Demonstration that CDC23's TPR domain directly contacts the destruction box of cyclin Clb2 and interacts with polo-like kinase revealed dual roles in substrate recognition and mitotic signaling.","evidence":"Yeast two-hybrid and in vitro binding with D-box mutants (Clb2); reciprocal Co-IP and genetic rescue of cut23-Plo1 interaction in fission yeast","pmids":["12413490","11777938"],"confidence":"Medium","gaps":["Substrate selectivity mechanism (why Clb2 but not other cyclins) unresolved","Whether the Plk and substrate-binding surfaces on the TPR domain overlap is unknown"]},{"year":2003,"claim":"Mass spectrometry-based phosphosite mapping showed that CDC23 is heavily phosphorylated by CDK1 during mitosis, and CDK1 phosphorylation suffices for Cdc20 binding, establishing the kinase-driven activation switch for the APC/C.","evidence":"MS phosphoproteomics on purified human APC/C; in vitro CDK1 and Plk1 kinase assays coupled to Cdc20 binding","pmids":["14657031"],"confidence":"High","gaps":["Contribution of individual CDC23 phosphosites versus other TPR subunits not dissected","In vivo validation of phosphosite requirements lacking"]},{"year":2011,"claim":"Functional knockdown in human cells demonstrated that CDC23 is required for cyclin B1 and securin turnover, and epistasis experiments confirmed these substrates mediate the cell cycle arrest phenotype.","evidence":"siRNA knockdown of CDC23 in thyroid cancer cells with flow cytometry and triple-knockdown rescue of proliferation","pmids":["21990323"],"confidence":"Medium","gaps":["Performed in a single cancer cell line","Whether CDC23 depletion affects APC/C integrity versus catalytic activity not distinguished"]},{"year":2016,"claim":"Determination of the crystal structure of the CDC23 N-terminal TPR domain provided the first atomic-resolution view of this APC/C scaffold subunit.","evidence":"X-ray crystallography at 3.1 Å using sulfur SAD phasing of human Cdc23 N-terminal domain","pmids":["26960127"],"confidence":"Medium","gaps":["Only N-terminal domain resolved; full-length structure in APC/C context not obtained at this resolution","No structure-function mutagenesis reported"]},{"year":2023,"claim":"Identification of CDC23 loss-of-function variants as a cause of human female infertility linked oocyte maturation defects to impaired APC/C-mediated securin and cyclin B1 degradation, with pharmacological rescue confirming the pathway.","evidence":"Patient homozygous missense variants (Y329C, R330C), Cdc23 knock-in mouse model, western blot for APC subunit levels, AZ3146 rescue","pmids":["37768355"],"confidence":"High","gaps":["Only two families studied","How decreased CDC23 protein level secondarily reduces APC4 not mechanistically explained"]},{"year":2024,"claim":"Biochemical reconstitution revealed that the CDC23 C-terminal disordered loop recruits Cks2 to position CDK-cyclin B for Apc1 autoinhibitory loop phosphorylation, defining the molecular logic of mitotic APC/C activation.","evidence":"Systematic loop-deletion mutagenesis of APC/C subunits with reconstituted activation assays","pmids":["38776225"],"confidence":"High","gaps":["Structural basis of Cks2 recruitment to the Apc8 loop not determined at atomic resolution","Whether this mechanism operates identically in oocytes versus somatic cells is untested"]},{"year":null,"claim":"The relative contributions of CDC23's multiple interaction surfaces (substrate D-box binding, Plk docking, Cks2 recruitment) to APC/C function in different cell types and developmental contexts remain to be dissected, and a complete high-resolution structure of the CDC23 C-terminal loop within the APC/C is lacking.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of the full-length CDC23 C-terminal disordered loop bound to Cks2","Cell-type-specific requirements for CDC23-mediated APC/C activation not systematically tested","Whether the Rac-CDC23 interaction has physiological relevance is unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,10,11]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[14]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,13]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4,8,9,11,14]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[10,14]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[13]}],"complexes":["APC/C"],"partners":["CDC20","CDC27","PLK1","CKS2","APC1","CLB2"],"other_free_text":[]},"mechanistic_narrative":"CDC23 (APC8) is a TPR-repeat-containing core structural subunit of the anaphase-promoting complex/cyclosome (APC/C), the essential E3 ubiquitin ligase that drives mitotic progression by targeting cyclins and securin for proteasomal degradation [PMID:2404612, PMID:9790767, PMID:18485873]. CDC23 undergoes CDK1-mediated mitotic phosphorylation that promotes Cdc20 binding and APC/C activation, and its C-terminal disordered loop recruits the CDK adaptor Cks2 to stimulate phosphorylation of the Apc1 autoinhibitory loop, synergizing with the Cdc27 loop for full activation [PMID:14657031, PMID:38776225]. The TPR domain of CDC23 mediates interaction with polo-like kinase required for metaphase-to-anaphase transition and directly contacts the destruction box of the mitotic cyclin Clb2, linking it to substrate recognition [PMID:11777938, PMID:12413490]. Homozygous missense variants in CDC23 (p.Y329C, p.R330C) cause female infertility through oocyte maturation arrest associated with reduced APC/C subunit levels and accumulation of securin and cyclin B1 [PMID:37768355]."},"prefetch_data":{"uniprot":{"accession":"Q9UJX2","full_name":"Cell division cycle protein 23 homolog","aliases":["Anaphase-promoting complex subunit 8","APC8","Cyclosome subunit 8"],"length_aa":597,"mass_kda":68.8,"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":"","url":"https://www.uniprot.org/uniprotkb/Q9UJX2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CDC23","classification":"Common 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SUBUNIT 10; ANAPC10","url":"https://www.omim.org/entry/613745"},{"mim_id":"609110","title":"F-BOX ONLY PROTEIN 43; FBXO43","url":"https://www.omim.org/entry/609110"},{"mim_id":"603462","title":"CELL DIVISION CYCLE 23; CDC23","url":"https://www.omim.org/entry/603462"},{"mim_id":"603461","title":"CELL DIVISION CYCLE 16; CDC16","url":"https://www.omim.org/entry/603461"}],"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/CDC23"},"hgnc":{"alias_symbol":["APC8","ANAPC8","CUT23"],"prev_symbol":[]},"alphafold":{"accession":"Q9UJX2","domains":[{"cath_id":"1.25.40.10","chopping":"298-396","consensus_level":"medium","plddt":94.3025,"start":298,"end":396},{"cath_id":"1.25.40.10","chopping":"397-507","consensus_level":"medium","plddt":87.5278,"start":397,"end":507},{"cath_id":"1.10.287","chopping":"522-566","consensus_level":"medium","plddt":80.3047,"start":522,"end":566}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJX2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJX2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJX2-F1-predicted_aligned_error_v6.png","plddt_mean":84.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CDC23","jax_strain_url":"https://www.jax.org/strain/search?query=CDC23"},"sequence":{"accession":"Q9UJX2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UJX2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UJX2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJX2"}},"corpus_meta":[{"pmid":"2404612","id":"PMC_2404612","title":"A 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regions punctuated by proline-induced turns, defining a new protein family required for mitosis.\",\n      \"method\": \"Sequence analysis and identification of repeating structural motif\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 computational/sequence analysis with functional context from genetic requirement; foundational structural discovery replicated across many subsequent studies\",\n      \"pmids\": [\"2404612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human CDC23 encodes a 591-amino acid TPR-containing protein that is a subunit of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for cyclin B ubiquitination and mitotic progression.\",\n      \"method\": \"cDNA cloning, genomic mapping, sequence homology analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — molecular cloning with direct sequence and structural characterization establishing APC/C subunit identity\",\n      \"pmids\": [\"9790767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"APC/C subunits Cdc23 and Cdc27 (S. cerevisiae) are required for the proteolysis of Cdc20 during S phase and early mitosis; during G1, Cdc20 destruction depends on its destruction box, but during S phase and mitosis Cdc23/Cdc27-dependent Cdc20 degradation is destruction-box-independent.\",\n      \"method\": \"Cell cycle-regulated protein stability assays, genetic analysis using cdc23 and cdc27 mutants\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with protein stability assays; multiple orthogonal methods in a single study\",\n      \"pmids\": [\"9651679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Fission yeast Cut23 (APC8/Cdc23 ortholog) is a subunit of the APC/cyclosome required for metaphase-to-anaphase transition; Cut23 protein levels fluctuate during the cell cycle and it may be ubiquitinated and degraded in a cell cycle-dependent fashion.\",\n      \"method\": \"Temperature-sensitive mutant screens, sucrose gradient centrifugation, metaphase arrest phenotype analysis\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic screen with biochemical fractionation establishing APC/C subunit identity and mitotic role\",\n      \"pmids\": [\"10526233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"S. cerevisiae CDC23 directly interacts with the nuclear protein SIN1 via its TPR repeat domain; the N-terminal domain of SIN1 binds the TPR region of CDC23 in vitro using recombinant proteins.\",\n      \"method\": \"Yeast two-hybrid assay and in vitro binding with recombinant proteins\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — two-hybrid plus in vitro pulldown with recombinant proteins; single lab\",\n      \"pmids\": [\"8710860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The destruction box (D box) of yeast mitotic cyclin Clb2 directly interacts with the APC/C subunit Cdc23; mutations in the Clb2 D box inhibited interaction with Cdc23 both in vivo (two-hybrid) and in vitro, providing the first evidence for direct APC/C substrate-subunit interaction.\",\n      \"method\": \"Yeast two-hybrid assay and in vitro binding assay with D box mutants\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — two-hybrid plus in vitro binding, with mutagenesis confirming specificity; single lab\",\n      \"pmids\": [\"12413490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Fission yeast polo kinase Plo1 physically interacts with APC/cyclosome through its noncatalytic polo-box domain and the TPR domain of Cut23 (APC8/Cdc23 ortholog); a cut23 mutation disrupting this interaction causes metaphase arrest that is rescued by Plo1 overexpression, indicating this interaction is required for mitotic progression.\",\n      \"method\": \"Co-immunoprecipitation, two-hybrid analysis, cut23 point mutant phenotypic analysis, genetic rescue\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, mutant with defined phenotype, genetic rescue; multiple orthogonal methods\",\n      \"pmids\": [\"11777938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Fission yeast Cdc23/Mcm10 is required for Cdc45 chromatin binding after pre-replicative complex (pre-RC) formation; inactivation of Cdc23 using degron mutations blocks Cdc45 chromatin association without affecting Mcm2 (pre-RC) binding.\",\n      \"method\": \"Cytological chromatin association assays, degron-based conditional inactivation, cell cycle staging\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional loss-of-function with specific molecular phenotype (Cdc45 vs Mcm2 chromatin binding) using orthogonal degron and cytological methods\",\n      \"pmids\": [\"12972571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Fission yeast Cdc23/Mcm10 stimulates phosphorylation of the Mcm complex (Mcm2 and Mcm4 subunits) by the Dfp1-Hsk1 kinase (DDK) by bridging the two complexes: Cdc23 binds selectively to Mcm467 subunits and to Dfp1, and its N-terminus is essential for Dfp1-Hsk1 interaction and for stimulating Mcm phosphorylation in vitro.\",\n      \"method\": \"In vitro kinase assay with purified components, co-immunoprecipitation, domain truncation analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstituted kinase assay with purified proteins plus domain mapping; multiple orthogonal methods\",\n      \"pmids\": [\"12604790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Fission yeast Cdc23/Mcm10 is required for the chromatin association and sub-nuclear distribution of DNA polymerase alpha-primase during S phase; TEV protease cleavage of Cdc23 or degron inactivation disrupts primase (Spp1) chromatin binding, but does not affect GINS complex (Psf2) chromatin association.\",\n      \"method\": \"TEV protease-mediated in vivo protein cleavage, degron allele inactivation, in situ chromatin binding assay, immunofluorescence\",\n      \"journal\": \"BMC molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two independent Cdc23 inactivation methods (TEV cleavage, degron) with specific molecular phenotype differentiating Polα-primase from GINS; moderate evidence\",\n      \"pmids\": [\"15941470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Activated Rac GTPases (Rac1, Rac2, Rac3) form a complex with the APC component CDC23 in mammalian cell lysates, suggesting that Rac may regulate specific APC-dependent proteolytic processes.\",\n      \"method\": \"Co-immunoprecipitation from mammalian cell lysates\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP, no functional follow-up of CDC23-Rac interaction\",\n      \"pmids\": [\"11445862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Knockdown of CDC23 in human thyroid cancer cells causes accumulation of cyclin B1 and securin and arrest in S and G2/M phases; triple knockdown of CDC23, cyclin B1, and securin reverses the proliferation and cell cycle defects, indicating CDC23's role in cell cycle progression is mediated at least in part through cyclin B1 and securin degradation.\",\n      \"method\": \"siRNA knockdown, cell cycle analysis by flow cytometry, triple knockdown epistasis, western blotting\",\n      \"journal\": \"Endocrine-related cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis by triple knockdown with defined substrate accumulation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21990323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The N-terminal domain of Cdc23 (APC/C subunit) was crystallized and its structure solved by sulfur SAD phasing to 3.1 Å resolution.\",\n      \"method\": \"X-ray crystallography (sulfur SAD phasing)\",\n      \"journal\": \"Acta crystallographica. Section D, Structural biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure determination; limited functional validation reported in this paper\",\n      \"pmids\": [\"26960127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Fission yeast Cdc23/Mcm10 is required for ribonucleotide imprint formation at the mating-type locus mat1; mutations in the Polα-recruitment and putative primase-homology domain of Mcm10/Cdc23 abrogate the alkali-labile ribonucleotide imprint, suggesting a direct role of Cdc23 in imprint installation in cooperation with Polα and Swi1.\",\n      \"method\": \"cdc23 allele mutant analysis, imprint detection by alkaline sensitivity assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific mutants with defined molecular phenotype (imprint formation); single lab\",\n      \"pmids\": [\"30759238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Homozygous missense variants in human CDC23 (p.Y329C and p.R330C) cause female infertility by disrupting oocyte maturation; p.Y329C reduces CDC23 protein level while p.R330C alters CDC23 subcellular localization in HeLa cells and mouse oocytes; knock-in mice (Cdc23Y329C/Y329C) accumulate securin and cyclin B1 in oocytes, and AZ3146 (MPS1 inhibitor) treatment rescues the phenotype.\",\n      \"method\": \"Patient variant identification, in vitro protein expression, subcellular localization assay, knock-in mouse model, western blotting for APC/C substrates, pharmacological rescue\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods: human variants, cellular localization, in vivo mouse model with substrate accumulation, pharmacological rescue; moderate-to-strong evidence\",\n      \"pmids\": [\"37768355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The C-terminal disordered loop domain of APC8 (Cdc23/Cut23 ortholog, Xenopus) directly recruits the CDK adaptor Xe-p9/Cks2, positioning the CDK-CyclinB complex near the autoinhibitory Apc1-300L loop to stimulate its phosphorylation and removal, thereby promoting APC/CCdc20 activation during mitosis; loss of both Apc8-L and Apc3-L renders APC/C inactive in mitosis.\",\n      \"method\": \"Loop-deletion mutant analysis, co-immunoprecipitation, in vitro APC/C activity assays, Xenopus egg extract system\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstituted Xenopus system with deletion mutants, co-IP, and functional activity assays; multiple orthogonal methods establishing mechanistic pathway\",\n      \"pmids\": [\"38776225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structures of S. cerevisiae APC/C reveal that, in contrast to human APC/C, the APC8 (Cdc23) subunit does not present a phospho-regulatable autoinhibitory segment blocking CDC20 C-box binding; instead the catalytic module is pre-positioned for E2 binding in the apo state, indicating species-specific regulatory differences in APC/C activation involving the APC8 subunit.\",\n      \"method\": \"Cryo-EM structural determination of S. cerevisiae APC/C in multiple states (apo, CDH1-substrate, phosphorylated)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures at medium resolution; preprint without peer review, but structural data is clear\",\n      \"pmids\": [\"bio_10.1101_2024.06.19.599685\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CDC23/APC8 is a core TPR-repeat-containing subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase that directly contacts APC/C substrates (via their destruction boxes), recruits polo kinase to the APC/C through its TPR domain, and contributes to APC/C mitotic activation through its C-terminal disordered loop domain (which recruits CDK-Cks adaptor to relieve autoinhibition); as the fission yeast ortholog Cdc23/Mcm10, it additionally functions in DNA replication by promoting Cdc45 chromatin loading, stimulating DDK (Dfp1-Hsk1) phosphorylation of the MCM complex, and facilitating DNA polymerase alpha-primase chromatin association.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1990,\n      \"finding\": \"CDC23 encodes a protein containing a novel repeating 34-amino acid motif (the TPR motif) reiterated several times; this motif defines a new family of proteins shared among genes required for mitosis and RNA synthesis, and is predicted to consist of amphipathic alpha-helical regions punctuated by proline-induced turns.\",\n      \"method\": \"Quantitative sequence analysis, cloning and characterization of CDC23 gene\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — foundational structural discovery, highly cited, replicated across multiple proteins\",\n      \"pmids\": [\"2404612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"The CDC23 gene of S. cerevisiae encodes a protein of 626 amino acids with four contiguous TPR repeats and a potential Ca2+-binding site near its N-terminus, expressed as a 2.4–2.5 kb transcript.\",\n      \"method\": \"Cloning, nucleotide sequencing, and ORF analysis\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct sequence characterization, single study\",\n      \"pmids\": [\"2205535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Yeast CDC23 directly interacts with the nuclear protein SIN1 (a transcriptional repressor involved in mitotic chromosome segregation); the N-terminus of SIN1 is sufficient to bind the TPR repeat domains of CDC23 in vitro.\",\n      \"method\": \"Yeast two-hybrid system, in vitro binding with recombinant proteins\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — two-hybrid plus in vitro binding, single lab\",\n      \"pmids\": [\"8710860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human CDC23 encodes a 591-amino acid, 68.3 kDa protein with 9 TPR units, mapping to chromosome 5q31; it is a component of the anaphase-promoting complex (APC/C), ubiquitously expressed, with 30% identity and 51% similarity to S. cerevisiae Cdc23.\",\n      \"method\": \"cDNA cloning, genomic mapping, sequence analysis, Northern blot\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — molecular cloning and characterization, single study\",\n      \"pmids\": [\"9790767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The APC/C subunits Cdc23 and Cdc27 are required for the degradation of the APC activator Cdc20 during S phase and early mitosis; this degradation occurs through a mechanism that does not depend on the Cdc20 destruction box, revealing a D-box-independent proteolytic pathway mediated by these APC components.\",\n      \"method\": \"Genetic analysis in S. cerevisiae, cell cycle protein stability assays, mutant analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis combined with biochemical stability assays, moderately cited\",\n      \"pmids\": [\"9651679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Fission yeast Cut23 (the S. pombe homolog of Cdc23/APC8) is identified as a subunit of the APC/cyclosome; it contains TPR repeats, its levels fluctuate during the cell cycle (possibly regulated by ubiquitination and degradation), and it is required for metaphase-anaphase progression. Cut23 interacts with polo kinase Plo1 through its TPR domain.\",\n      \"method\": \"Temperature-sensitive mutant screen, sucrose gradient centrifugation, genetic analysis\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — biochemical fractionation plus genetic analysis, single study\",\n      \"pmids\": [\"10526233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The small GTPase Rac forms a complex with the APC component CDC23 in mammalian cell lysates, suggesting that activated Rac may regulate APC/C function or subcellular localization through direct interaction with CDC23.\",\n      \"method\": \"Co-immunoprecipitation from mammalian cell lysates, yeast two-hybrid\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP, limited mechanistic follow-up\",\n      \"pmids\": [\"11445862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The APC/C subunit Cdc23 directly interacts with the destruction box (D-box) of the mitotic cyclin Clb2, but not with D-boxes of Clb1, Clb3, or Clb5; mutations in the Clb2 D-box inhibit interaction with Cdc23 both in vivo and in vitro, providing the first evidence for direct APC/C substrate recognition by a specific APC subunit.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, D-box mutant analysis\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — two-hybrid plus in vitro binding with mutagenesis, single lab\",\n      \"pmids\": [\"12413490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Fission yeast polo-like kinase Plo1 physically interacts with the APC/cyclosome through the noncatalytic polo-box domain of Plo1 and the TPR domain of Cut23 (APC8/Cdc23 homolog); a cut23 mutation that specifically disrupts the Plo1 interaction causes metaphase arrest that can be rescued by high Plo1 expression, indicating this interaction is required for mitotic progression.\",\n      \"method\": \"Co-immunoprecipitation, genetic analysis with cut23 separation-of-function mutation, rescue by Plo1 overexpression\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus genetic rescue with defined phenotype, moderately cited\",\n      \"pmids\": [\"11777938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Mitotic phosphorylation of the human APC/C occurs on at least 43 sites, with 32 sites clustered in Apc1 and the TPR subunits Cdc27, Cdc16, Cdc23, and Apc7; at least 15 mitosis-specific sites are generated by CDK1 and 3 by Plk1 in vitro. CDK1-mediated APC phosphorylation (but not Plk1) is sufficient for increased Cdc20 binding and APC activation.\",\n      \"method\": \"Mass spectrometry phosphoproteomics, in vitro kinase assays with CDK1 and Plk1, Cdc20 binding assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay plus mass spectrometry phosphosite mapping, highly cited\",\n      \"pmids\": [\"14657031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The human APC/C assembles K11-linked ubiquitin chains on substrates; the TEK-box surface of ubiquitin, homologous to motifs in APC/C substrates, enables the APC/C to switch from substrate lysines to ubiquitin lysines during chain elongation. CDC23 (as part of the APC/C) participates in this ubiquitin chain assembly mechanism.\",\n      \"method\": \"In vitro ubiquitination assays, ubiquitin chain topology analysis, mutagenesis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro ubiquitination with mutagenesis, highly cited\",\n      \"pmids\": [\"18485873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Functional knockdown of CDC23 in human thyroid cancer cells causes accumulation of cells in S and G2/M phases, inhibits proliferation, tumor spheroid formation, and anchorage-independent growth, with significant accumulation of cyclin B1 and securin protein; triple knockdown of CDC23, cyclin B1, and securin reverses the proliferation and cell cycle effects, placing CDC23 upstream of cyclin B1 and securin degradation.\",\n      \"method\": \"siRNA knockdown, cell cycle analysis by flow cytometry, western blotting, triple knockdown rescue experiment, in vitro proliferation and colony assays\",\n      \"journal\": \"Endocrine-related cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined phenotype and epistasis rescue, single lab\",\n      \"pmids\": [\"21990323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The crystal structure of the N-terminal domain of human Cdc23 (Cdc23(Nterm)), a subunit of the APC/C, was determined by sulfur SAD phasing to 3.1 Å resolution, providing structural insight into the TPR-containing APC/C subunit.\",\n      \"method\": \"X-ray crystallography, sulfur SAD phasing\",\n      \"journal\": \"Acta crystallographica. Section D, Structural biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure determination, limited functional validation in this paper\",\n      \"pmids\": [\"26960127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Homozygous missense variants in CDC23 (p.Y329C and p.R330C) cause female infertility with oocyte maturation defects; p.Y329C decreases CDC23 protein level, while p.R330C alters CDC23 localization in HeLa cells and mouse oocytes. Knock-in mice (Cdc23Y329C/Y329C) show low CDC23 and APC4 levels and accumulation of securin and cyclin B1 in oocytes; treatment with AZ3146 (a Mps1 kinase inhibitor) rescues the phenotype.\",\n      \"method\": \"Patient variant identification, in vitro expression assays, immunofluorescence localization, knock-in mouse model, western blotting, pharmacological rescue\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including mouse knock-in, localization, biochemical analysis, and pharmacological rescue\",\n      \"pmids\": [\"37768355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The C-terminal disordered loop of APC8 (Cdc23 homolog) directly recruits the CDK adaptor protein Xe-p9/Cks2, positioning the CDK-cyclin B complex near the autoinhibitory Apc1-300L loop; this stimulates phosphorylation and removal of Apc1-300L to form active APC/C-Cdc20. Apc8-L acts synergistically with Apc3-L (Cdc27 loop) for full mitotic APC/C activation.\",\n      \"method\": \"Systematic loop-deletion mutant analysis of APC/C, biochemical reconstitution, protein interaction assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstituted biochemistry with systematic mutagenesis of loop domains, mechanistic validation\",\n      \"pmids\": [\"38776225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Comparative cryo-EM structures of S. cerevisiae APC/C reveal that APC8 (Cdc23) contributes to overall scaffold architecture; unlike in human APC/C where the CDC20 C-box binding site of APC8 is sterically blocked by the phospho-regulatable Apc1 autoinhibitory segment, S. cerevisiae APC/C shows no evidence of this autoinhibitory segment blocking APC8, indicating species-specific differences in coactivator-mediated APC/C activation mechanisms.\",\n      \"method\": \"Cryo-EM structure determination of multiple S. cerevisiae APC/C complexes, comparative structural analysis with human APC/C\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures, preprint without peer review\",\n      \"pmids\": [\"bio_10.1101_2024.06.19.599685\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CDC23 (human APC8) is a TPR-repeat-containing core subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase that undergoes CDK1-mediated mitotic phosphorylation to enable Cdc20 binding and APC/C activation; its C-terminal disordered loop recruits CDK adaptor Cks2 to stimulate phosphorylation of the Apc1 autoinhibitory loop, its TPR domain mediates interaction with polo-like kinase for mitotic progression, it directly contacts the destruction box of substrates such as cyclin Clb2, it controls degradation of Cdc20 and the accumulation of cyclin B1 and securin, and loss-of-function variants cause female infertility through oocyte maturation defects associated with securin and cyclin B1 accumulation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CDC23 (APC8) is a TPR-repeat-containing core subunit of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase, essential for mitotic progression through regulated ubiquitin-dependent degradation of cell-cycle substrates including cyclin B1, securin, and the APC/C co-activator Cdc20 [PMID:9651679, PMID:21990323, PMID:37768355]. CDC23 directly contacts APC/C substrates via their destruction box motifs and recruits polo kinase through its TPR domain, an interaction required for the metaphase-to-anaphase transition [PMID:12413490, PMID:11777938]. The C-terminal disordered loop of CDC23 recruits the CDK-Cks adaptor to stimulate phosphorylation-dependent relief of APC/C autoinhibition, thereby promoting APC/C^Cdc20 activation in mitosis [PMID:38776225]. Homozygous missense variants in human CDC23 (p.Y329C, p.R330C) cause female infertility through defective oocyte maturation, with accumulation of APC/C substrates securin and cyclin B1 confirmed in a knock-in mouse model [PMID:37768355].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Identification of CDC23 as a TPR-repeat protein established a new structural motif family linked to mitotic function, answering what structural features define this essential cell-cycle gene.\",\n      \"evidence\": \"Sequence analysis of S. cerevisiae CDC23 identifying the 34-amino acid tetratricopeptide repeat\",\n      \"pmids\": [\"2404612\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical function assigned to the TPR repeats\", \"No mammalian ortholog characterized\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing CDC23 as an APC/C subunit in both yeast and humans unified its genetic requirement for mitosis with a specific E3 ligase complex, and revealed that APC/C subunits Cdc23/Cdc27 mediate cell-cycle-dependent proteolysis of the co-activator Cdc20 through both D-box-dependent and -independent mechanisms.\",\n      \"evidence\": \"Human CDC23 cDNA cloning plus S. cerevisiae cdc23/cdc27 mutant protein stability assays across cell cycle stages\",\n      \"pmids\": [\"9790767\", \"9651679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of D-box-independent Cdc20 degradation unclear\", \"Direct substrate contacts not yet demonstrated\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Two key interaction surfaces of CDC23 were defined: direct contact with cyclin destruction boxes established CDC23 as a substrate-recognition subunit, and polo kinase recruitment via the TPR domain linked CDC23 to mitotic kinase signaling required for the metaphase-to-anaphase transition.\",\n      \"evidence\": \"Yeast two-hybrid and in vitro binding with D-box mutants (S. cerevisiae); co-IP, two-hybrid, and cut23 point mutant rescue by Plo1 overexpression (S. pombe)\",\n      \"pmids\": [\"12413490\", \"11777938\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of D-box recognition by CDC23 unresolved\", \"Whether polo kinase–CDC23 interaction is conserved in vertebrates not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The fission yeast ortholog Cdc23/Mcm10 was shown to function in DNA replication initiation by promoting Cdc45 chromatin loading and bridging the DDK kinase to the MCM helicase to stimulate MCM phosphorylation — establishing a replication role distinct from APC/C function.\",\n      \"evidence\": \"Degron-based conditional inactivation with chromatin binding assays; in vitro reconstituted kinase assay with purified Cdc23, MCM, and Dfp1-Hsk1\",\n      \"pmids\": [\"12972571\", \"12604790\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this replication function is conserved in metazoans (where Mcm10 and APC8 are separate genes) remains unclear\", \"Structural basis of Cdc23-MCM and Cdc23-Dfp1 interactions not determined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"CDC23/Mcm10 was further shown to recruit DNA polymerase α-primase to chromatin during S phase, separating this function from GINS complex loading and extending CDC23's replication roles beyond helicase activation.\",\n      \"evidence\": \"TEV protease-mediated in vivo cleavage and degron inactivation of Cdc23 with differential chromatin binding of Spp1 versus Psf2 in S. pombe\",\n      \"pmids\": [\"15941470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Pol α recruitment by Cdc23 requires direct physical interaction not conclusively shown\", \"In vitro reconstitution of Pol α recruitment lacking\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"siRNA depletion of CDC23 in human cells demonstrated that its APC/C function in cyclin B1 and securin degradation is rate-limiting for cell cycle progression, as triple knockdown of CDC23 with its substrates rescued proliferation defects.\",\n      \"evidence\": \"siRNA knockdown and triple epistasis in human thyroid cancer cells with flow cytometry and western blotting\",\n      \"pmids\": [\"21990323\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cancer cell line context limits generalizability\", \"Off-target siRNA effects not fully controlled\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Human CDC23 missense variants were identified as a cause of female infertility due to defective oocyte maturation, directly linking APC/C substrate accumulation (securin, cyclin B1) to a Mendelian reproductive phenotype and validating CDC23 function in human meiosis.\",\n      \"evidence\": \"Patient variant identification, HeLa and mouse oocyte localization assays, Cdc23 Y329C knock-in mouse model with APC/C substrate accumulation, MPS1 inhibitor rescue\",\n      \"pmids\": [\"37768355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether R330C variant affects APC/C complex integrity versus substrate recognition not resolved\", \"Mechanism by which MPS1 inhibition bypasses CDC23 deficiency not fully explained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The C-terminal disordered loop of CDC23 was shown to recruit CDK-Cks to the APC/C, enabling phosphorylation-dependent removal of the Apc1 autoinhibitory loop — establishing CDC23 as a direct mediator of mitotic APC/C activation rather than merely a structural scaffold.\",\n      \"evidence\": \"Loop-deletion mutants, co-IP, and in vitro APC/C activity assays in Xenopus egg extracts\",\n      \"pmids\": [\"38776225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the loop-based CDK-Cks recruitment mechanism operates identically in mammalian APC/C\", \"Structural details of the CDC23 loop–Cks interface not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the high-resolution structural basis for how CDC23 simultaneously contacts substrates, polo kinase, and CDK-Cks; whether polo kinase recruitment through the CDC23 TPR domain is conserved in vertebrates; and the precise mechanism by which CDC23 disease variants impair APC/C function in human meiosis.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of full-length CDC23 in complex with substrates or regulatory kinases\", \"Vertebrate polo kinase–CDC23 interaction not directly tested\", \"Mechanistic basis of CDC23 variant pathogenicity in human oocytes incompletely defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 6, 8, 15]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 11, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4, 14]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [7, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 6, 11, 14, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 5, 11, 15]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [7, 8, 9]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"complexes\": [\n      \"APC/C\"\n    ],\n    \"partners\": [\n      \"CDC27\",\n      \"PLO1\",\n      \"CDC45\",\n      \"DFP1\",\n      \"MCM4\",\n      \"CKS2\",\n      \"CDC20\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CDC23 (APC8) is a TPR-repeat-containing core structural subunit of the anaphase-promoting complex/cyclosome (APC/C), the essential E3 ubiquitin ligase that drives mitotic progression by targeting cyclins and securin for proteasomal degradation [PMID:2404612, PMID:9790767, PMID:18485873]. CDC23 undergoes CDK1-mediated mitotic phosphorylation that promotes Cdc20 binding and APC/C activation, and its C-terminal disordered loop recruits the CDK adaptor Cks2 to stimulate phosphorylation of the Apc1 autoinhibitory loop, synergizing with the Cdc27 loop for full activation [PMID:14657031, PMID:38776225]. The TPR domain of CDC23 mediates interaction with polo-like kinase required for metaphase-to-anaphase transition and directly contacts the destruction box of the mitotic cyclin Clb2, linking it to substrate recognition [PMID:11777938, PMID:12413490]. Homozygous missense variants in CDC23 (p.Y329C, p.R330C) cause female infertility through oocyte maturation arrest associated with reduced APC/C subunit levels and accumulation of securin and cyclin B1 [PMID:37768355].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Identification of the TPR motif in CDC23 established a structural framework for understanding how a repetitive α-helical fold could underpin protein-protein interactions in cell cycle regulators.\",\n      \"evidence\": \"Quantitative sequence analysis and cloning of yeast CDC23 revealing iterated 34-amino acid repeats\",\n      \"pmids\": [\"2404612\", \"2205535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional assay linking TPR repeats to APC/C activity\", \"Mammalian homolog not yet cloned\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Cloning of human CDC23 and its assignment as an APC/C subunit, combined with genetic evidence that Cdc23 controls Cdc20 degradation, placed CDC23 within the ubiquitin-dependent cell cycle machinery.\",\n      \"evidence\": \"cDNA cloning and Northern blot of human CDC23; genetic stability assays in S. cerevisiae showing Cdc23/Cdc27-dependent, D-box-independent Cdc20 turnover\",\n      \"pmids\": [\"9790767\", \"9651679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of D-box-independent Cdc20 degradation unknown\", \"Physical interaction between CDC23 and substrates not yet shown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstration that CDC23's TPR domain directly contacts the destruction box of cyclin Clb2 and interacts with polo-like kinase revealed dual roles in substrate recognition and mitotic signaling.\",\n      \"evidence\": \"Yeast two-hybrid and in vitro binding with D-box mutants (Clb2); reciprocal Co-IP and genetic rescue of cut23-Plo1 interaction in fission yeast\",\n      \"pmids\": [\"12413490\", \"11777938\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate selectivity mechanism (why Clb2 but not other cyclins) unresolved\", \"Whether the Plk and substrate-binding surfaces on the TPR domain overlap is unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mass spectrometry-based phosphosite mapping showed that CDC23 is heavily phosphorylated by CDK1 during mitosis, and CDK1 phosphorylation suffices for Cdc20 binding, establishing the kinase-driven activation switch for the APC/C.\",\n      \"evidence\": \"MS phosphoproteomics on purified human APC/C; in vitro CDK1 and Plk1 kinase assays coupled to Cdc20 binding\",\n      \"pmids\": [\"14657031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contribution of individual CDC23 phosphosites versus other TPR subunits not dissected\", \"In vivo validation of phosphosite requirements lacking\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Functional knockdown in human cells demonstrated that CDC23 is required for cyclin B1 and securin turnover, and epistasis experiments confirmed these substrates mediate the cell cycle arrest phenotype.\",\n      \"evidence\": \"siRNA knockdown of CDC23 in thyroid cancer cells with flow cytometry and triple-knockdown rescue of proliferation\",\n      \"pmids\": [\"21990323\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Performed in a single cancer cell line\", \"Whether CDC23 depletion affects APC/C integrity versus catalytic activity not distinguished\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Determination of the crystal structure of the CDC23 N-terminal TPR domain provided the first atomic-resolution view of this APC/C scaffold subunit.\",\n      \"evidence\": \"X-ray crystallography at 3.1 Å using sulfur SAD phasing of human Cdc23 N-terminal domain\",\n      \"pmids\": [\"26960127\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only N-terminal domain resolved; full-length structure in APC/C context not obtained at this resolution\", \"No structure-function mutagenesis reported\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of CDC23 loss-of-function variants as a cause of human female infertility linked oocyte maturation defects to impaired APC/C-mediated securin and cyclin B1 degradation, with pharmacological rescue confirming the pathway.\",\n      \"evidence\": \"Patient homozygous missense variants (Y329C, R330C), Cdc23 knock-in mouse model, western blot for APC subunit levels, AZ3146 rescue\",\n      \"pmids\": [\"37768355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Only two families studied\", \"How decreased CDC23 protein level secondarily reduces APC4 not mechanistically explained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Biochemical reconstitution revealed that the CDC23 C-terminal disordered loop recruits Cks2 to position CDK-cyclin B for Apc1 autoinhibitory loop phosphorylation, defining the molecular logic of mitotic APC/C activation.\",\n      \"evidence\": \"Systematic loop-deletion mutagenesis of APC/C subunits with reconstituted activation assays\",\n      \"pmids\": [\"38776225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Cks2 recruitment to the Apc8 loop not determined at atomic resolution\", \"Whether this mechanism operates identically in oocytes versus somatic cells is untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The relative contributions of CDC23's multiple interaction surfaces (substrate D-box binding, Plk docking, Cks2 recruitment) to APC/C function in different cell types and developmental contexts remain to be dissected, and a complete high-resolution structure of the CDC23 C-terminal loop within the APC/C is lacking.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of the full-length CDC23 C-terminal disordered loop bound to Cks2\", \"Cell-type-specific requirements for CDC23-mediated APC/C activation not systematically tested\", \"Whether the Rac-CDC23 interaction has physiological relevance is unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 10, 11]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 8, 9, 11, 14]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [10, 14]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"complexes\": [\"APC/C\"],\n    \"partners\": [\"CDC20\", \"CDC27\", \"PLK1\", \"CKS2\", \"APC1\", \"CLB2\"],\n    \"other_free_text\": []\n  }\n}\n```"}