{"gene":"CDC6","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1989,"finding":"CDC6 gene encodes an essential protein for S phase entry in S. cerevisiae; the deduced protein sequence contains a conserved nucleotide-binding site, and disruption of CDC6 is lethal for mitotic growth.","method":"Complementation cloning, gene disruption, DNA sequencing","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation and gene disruption with defined lethal phenotype, single study","pmids":["2656692"],"is_preprint":false},{"year":1990,"finding":"CDC6 mRNA is periodically expressed in the yeast cell cycle, peaking at the G1/S boundary, and the CDC6 promoter contains sequence elements (similar to those in other cell cycle-regulated genes) that drive this periodic transcription.","method":"Synchronized culture experiments (alpha-factor arrest, elutriation), Northern blotting, promoter sequence analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent synchronization methods, single lab","pmids":["2246267"],"is_preprint":false},{"year":1992,"finding":"Constitutive CDC6 expression in yeast delays initiation of M phase in a manner dependent on the Wee1/Mik1 mitotic inhibitor kinases and is counteracted by Cdc25/MIH1 phosphatases, indicating CDC6 indirectly inhibits activation of p34cdc2/CDC28 M-phase kinase; CDC6 thus has a dual role in requiring DNA replication initiation and suppressing nuclear division.","method":"Constitutive expression of CDC6 in budding and fission yeast, genetic interaction analysis with wee1, mik1, cdc25, MIH1 mutants","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in two yeast species, single lab","pmids":["1600944"],"is_preprint":false},{"year":1994,"finding":"Purified S. cerevisiae Cdc6 protein binds rATP and rGTP upon UV cross-linking and catalyzes DNA-independent hydrolysis of purine nucleoside triphosphates, consistent with an ATPase/GTPase activity that may regulate replication initiation.","method":"Recombinant protein expression in E. coli, UV cross-linking nucleotide binding assay, ATPase/GTPase activity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assay with purified protein, single lab, no mutagenesis reported","pmids":["8083240"],"is_preprint":false},{"year":1995,"finding":"Cdc6 is an unstable protein whose de novo synthesis in G1 (driven first by Swi5, then by MBF/SBF transcription factors) is required for initiation of DNA replication; cells lacking Cdc6 fail to replicate DNA but still undergo mitosis ('reductional anaphase'), demonstrating that Cdc6 deficiency uncouples DNA replication from mitotic entry.","method":"Yeast genetics, cell synchronization, fluorescence in situ hybridization (FISH), transcription factor mutant analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic approaches and FISH in budding yeast, replicated across conditions","pmids":["7641697"],"is_preprint":false},{"year":1996,"finding":"ORC and Cdc6 form distinct chromatin complexes at replication origins in S. cerevisiae: origins oscillate between an ORC-dependent post-replicative state and a Cdc6-dependent pre-replicative state during the cell cycle.","method":"Genomic footprinting at single-copy chromosomal origins in yeast","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct in vivo footprinting at endogenous loci, multiple origins tested","pmids":["8978693"],"is_preprint":false},{"year":1996,"finding":"Yeast Cdc6 physically interacts with B-type cyclin/Cdc28 kinase complexes (not Cln/Cdc28); Cdc6 is a substrate and inhibitor of Cdc28 kinase in vitro; the Cdc28-interaction domain of Cdc6 is required for its essential growth function and for restraining mitosis.","method":"Co-immunoprecipitation, p13-agarose pulldown, affinity chromatography with bacterially produced Cdc6, in vitro kinase assay, deletion mutant analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reciprocal pulldown, in vitro kinase assay, and genetic complementation with deletion mutant, single lab","pmids":["8930895"],"is_preprint":false},{"year":1998,"finding":"Human CDC6 transcription is regulated by E2F transcription factors; E2F binding sites in the CDC6 promoter are required for cell cycle-regulated expression; microinjection of anti-CDC6 antibody blocks initiation of DNA replication in human tumor cells.","method":"Promoter-reporter assay, in vivo footprinting, microinjection of antibody, E2F overexpression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (promoter analysis, in vivo footprinting, antibody microinjection), replicated by independent labs","pmids":["9520412"],"is_preprint":false},{"year":1998,"finding":"Human CDC6/Cdc18 is nuclear in G1 and is selectively eliminated from the nucleus at the onset of S phase; it associates with human Orc1 protein and cyclin-CDK complexes; nuclear localization is independent of its nuclear localization signal, implying association with other nuclear proteins.","method":"Epitope-tagged protein cell cycle fractionation, co-immunoprecipitation with Orc1 and cyclin-CDKs, site-directed mutagenesis of NLS","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, subcellular fractionation, NLS mutagenesis; multiple orthogonal methods in single study","pmids":["9566895"],"is_preprint":false},{"year":1998,"finding":"Mammalian CDC6 promoter is activated by E2F proteins; E2F binding sites are required for serum-stimulated and cell cycle-regulated expression; CDC6 cooperates with cyclin E to induce S phase entry; microinjection of anti-CDC6 antiserum blocks DNA synthesis.","method":"Promoter-reporter assay, E2F overexpression, co-transfection with cyclin E, antibody microinjection","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, consistent with independent reports","pmids":["9774682"],"is_preprint":false},{"year":1998,"finding":"Recombinant human Cdc6 (HsCdc6) specifically binds ATP and slowly hydrolyzes it; Walker A and B motif mutants are defective in ATP binding/hydrolysis and display aberrant conformations in the presence of nucleotides; microinjection of either mutant inhibits DNA replication in G1 cells, demonstrating that Cdc6 ATPase activity is essential for DNA replication.","method":"Recombinant protein expression, ATP binding and hydrolysis assays, mutagenesis of Walker A and B motifs, microinjection into human cells","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis plus in vivo functional validation, single lab","pmids":["10436018"],"is_preprint":false},{"year":1998,"finding":"Cdc6 protein causes premature entry into S phase: addition of recombinant Cdc6 to permeabilized G1 nuclei induces up to 82% of nuclei to initiate DNA replication and accelerates G1 progression in a mammalian cell-free system; quiescent cells lack Cdc6 and fail to load MCM proteins onto chromatin.","method":"Mammalian cell-free DNA replication system, recombinant Cdc6 addition, immunoblot for Cdc6 and MCM chromatin association","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution in cell-free system with quantitative readout, single lab","pmids":["9857179"],"is_preprint":false},{"year":1999,"finding":"Mammalian CDC6 is phosphorylated specifically by Cyclin A/CDK2 (not Cyclin E or Cyclin B) via an N-terminal Cy-motif; phosphorylation of three N-terminal CDK consensus sites regulates CDC6 subcellular localization; CDC6 is nuclear in G1 and relocalizes to the cytoplasm upon Cyclin A/CDK2 activation, suggesting phosphorylation prevents re-replication.","method":"In vitro kinase assay, co-immunoprecipitation, cyclin binding domain mapping, ectopic Cyclin A/E expression, subcellular fractionation and immunofluorescence","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay plus in vivo co-IP, domain mapping, and localization experiments; multiple orthogonal methods","pmids":["9889196"],"is_preprint":false},{"year":1999,"finding":"S. cerevisiae Cdc6 protein is ubiquitinated in vivo and degraded by a Cdc4-dependent (SCF) ubiquitin-mediated proteolytic pathway at the late G1/early S phase transition.","method":"In vivo ubiquitination assay, analysis of Cdc6 stability in cdc4 mutants, cell cycle synchronization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo ubiquitination detection and genetic epistasis with cdc4, single lab","pmids":["10085159"],"is_preprint":false},{"year":1999,"finding":"After formation of pre-initiation complexes (ORC, Cdc6, MCM on chromatin), Cdc6 is rapidly removed from chromatin in Xenopus extracts, possibly via a cdk2-activated ubiquitin-dependent proteolytic pathway; inhibition of this removal blocks DNA replication initiation; subsequent initiation steps are independent of ORC and Cdc6 but dependent on cdk2 activity.","method":"Xenopus laevis egg extract system, chromatin fractionation, cdk2 inhibition, ubiquitin pathway interference","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted cell-free system with defined biochemical perturbations, multiple epistasis tests in single study","pmids":["9442103"],"is_preprint":false},{"year":2000,"finding":"Human CDC6 is targeted for ubiquitin-mediated proteolysis by APC/C-CDH1 in G1 and quiescent cells; point mutations in both the destruction box and KEN-box motifs together stabilize CDC6; APC/CDH1 ubiquitinates CDC6 in vitro; APC and CDH1 are required and limiting for CDC6 proteolysis in vivo.","method":"In vitro ubiquitination assay with purified APC/CDH1, APC/CDH1 depletion, mutagenesis of destruction box and KEN box, stability assays in G1 and quiescent cells","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution of ubiquitination plus in vivo depletion with mutagenesis; multiple orthogonal methods","pmids":["10995389"],"is_preprint":false},{"year":2000,"finding":"Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a process requiring nuclear import and phosphorylation by a chromatin-bound kinase; recombinant Cyclin A-CDK2 completely substitutes for the nucleus in promoting destruction of soluble Cdc6, suggesting that Cyclin A-CDK2 phosphorylation destroys free Cdc6 not assembled into replication complexes.","method":"Mammalian cell extract in vitro replication system, chromatin fractionation, recombinant Cyclin A-CDK2 addition, nuclear import inhibition","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — cell-free biochemical reconstitution with defined kinase substitution, single lab","pmids":["10806104"],"is_preprint":false},{"year":2000,"finding":"Cdc6 stability in S. cerevisiae is regulated by Cdc28 (CDK1)/Clb kinase activity; Cdc6 mutants lacking Cdc28 phosphorylation sites are stabilized; loss of Cdc28/Clb kinase activity allows accumulation of Cdc6 protein in mitotic-arrested cells.","method":"Cell cycle synchronization, protein stability assays, phosphorylation-site mutant analysis, temperature-sensitive cdc28 mutants","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation-site mutagenesis and genetic epistasis, single lab","pmids":["10734126"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of an archaeal Cdc6 ortholog (2.0 Å) reveals an AAA+-type nucleotide binding fold bound to Mg·ADP and a winged-helix (WH) domain similar to known DNA-binding modules; mutagenesis of the WH domain of S. pombe Cdc18 shows this region is required for function in vivo; nucleotide binding/hydrolysis by Cdc6/Cdc18 is required for S phase progression and for maintenance of S-phase checkpoint control.","method":"X-ray crystallography (2.0 Å), site-directed mutagenesis of WH domain and ATPase motifs, in vivo functional assays in S. pombe","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure combined with mutagenesis and in vivo functional validation","pmids":["11030343"],"is_preprint":false},{"year":2001,"finding":"Cyclin E binds the N-terminal region of Cdc6 via RXL (Cy) motifs on Cdc6 and the substrate-selection (MRAIL) motif on Cyclin E; this interaction localizes Cyclin E-CDK2 to chromatin in an ORC- and Cdc6-dependent manner; mutation of the RXL motifs in Cdc6 abrogates Cyclin E binding and rescuing of replication in Cdc6-depleted Xenopus extracts.","method":"Xenopus egg extract chromatin assembly assay, domain mapping, site-directed mutagenesis of RXL and MRAIL motifs, rescue assays with Cdc6-depleted extracts","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis of both interacting proteins and rescue experiments","pmids":["11257126"],"is_preprint":false},{"year":2001,"finding":"Cdc6 expression in fission yeast G2 cells overrides controls that ensure one S phase per cycle by re-firing replication origins and recruiting MCM Cdc21 to chromatin; co-expression of Cdt1 greatly amplifies this re-replication; Cdt1 may stabilize Cdc18 on chromatin.","method":"Inducible expression in S. pombe G2 cells, FACS analysis, MCM chromatin association, phosphorylation-site mutant analysis","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic inducible system with chromatin association readout, single lab","pmids":["11532929"],"is_preprint":false},{"year":2001,"finding":"Cdc6 cooperates with Sic1 to inactivate mitotic CDKs during late mitosis in S. cerevisiae; deletion of the CDK-interacting domain of Cdc6 causes a mitotic exit delay that is accentuated in the absence of Sic1 or cyclin degradation; Cdc6, like Sic1, binds CDK complexes in vivo and downregulates them in vitro.","method":"Genetic deletion of Cdc6 CDK-binding domain, double mutant analysis with sic1 and cyclin degradation mutants, co-immunoprecipitation, in vitro CDK kinase assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro CDK inhibition, in vivo co-IP, and epistasis with multiple genetic backgrounds, single lab with multiple orthogonal methods","pmids":["11460169"],"is_preprint":false},{"year":2002,"finding":"Human Cdc6 is rapidly destroyed by a proteasome- and ubiquitin-dependent pathway during early apoptosis induced by adozelesin (p53-independent) and by a separate caspase-dependent pathway during TNF-α-induced apoptosis; the proteasome-dependent pathway is conserved in S. cerevisiae.","method":"Western blot of Cdc6 during apoptosis, proteasome inhibitor, p53-null cell lines, cross-species comparison","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological pathway dissection in human and yeast cells, single lab","pmids":["12006651"],"is_preprint":false},{"year":2002,"finding":"Mammalian Cdc6 expression alone (via adenoviral vector) is sufficient to induce stable MCM chromatin association in quiescent cells with low CDK activity; Cdc6 ATP-binding site mutation severely impairs MCM loading; Cdc6 synergizes with cyclin E/CDK2 (but not cyclin A/CDK2) to induce semiconservative DNA replication in quiescent cells.","method":"Adenoviral expression of wild-type and mutant Cdc6 in quiescent cells, chromatin fractionation for MCM, semiconservative replication assay, CDK co-expression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional dissection with ATPase mutants, CDK specificity determination, and MCM loading assay in intact mammalian cells","pmids":["11805305"],"is_preprint":false},{"year":2002,"finding":"Xenopus Cdc6 synthesis during meiosis I (at germinal vesicle breakdown, GVBD) is necessary and sufficient for re-establishing DNA replication competence in oocytes; injection of Cdc6 protein into GVBD oocytes induces DNA replication in the absence of other protein synthesis.","method":"Xenopus oocyte injection of recombinant Cdc6 protein, replication competence assay, protein synthesis inhibition","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution by direct protein injection with protein synthesis inhibitor control, single lab","pmids":["12384699"],"is_preprint":false},{"year":2002,"finding":"Human Cdc6 is specifically cleaved by caspase-3 during apoptosis; expression of a caspase-uncleavable Cdc6 mutant attenuates apoptosis, demonstrating that Cdc6 cleavage facilitates cell death and prevents a wounded cell from replicating.","method":"Western blot of Cdc6 cleavage during apoptosis in multiple cell lines, expression of cleavage-resistant Cdc6 mutant, apoptosis assays","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — caspase-3 cleavage site identification with dominant-negative mutant, single lab","pmids":["12151338"],"is_preprint":false},{"year":2003,"finding":"Caspase-3 cleaves Cdc6 at SEVD442/G site during apoptosis, generating p49-tCdc6 that lacks the C-terminal nuclear export sequence; p49-tCdc6 is retained in the nucleus (resistant to Cyclin A-CDK2-mediated export), acts as a dominant negative inhibitor of DNA replication, and promotes apoptosis when ectopically expressed.","method":"In vitro caspase-3 cleavage assay, mutagenesis of cleavage sites, subcellular fractionation, ectopic expression of truncated Cdc6, apoptosis assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical cleavage site mapping with functional nuclear retention and apoptosis assays, single lab","pmids":["14517333"],"is_preprint":false},{"year":2003,"finding":"Human Cdc6 overexpression in G2 phase prevents entry into mitosis via a Chk1-dependent checkpoint; this block is abolished by constitutively active Cyclin B/CDK1, Cdc25B, or Cdc25C, or by the Chk1 inhibitor UCN-01; overexpressed Cdc6 in G2 induces Chk1 phosphorylation.","method":"Ectopic HuCdc6 overexpression in G2-phase cells, kinase inhibitor (UCN-01), Cdc25 co-expression, flow cytometry, Chk1 phosphorylation assay","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological epistasis plus genetic co-expression with defined readout, single lab","pmids":["12554670"],"is_preprint":false},{"year":2003,"finding":"Biochemical characterization of archaeal Cdc6 (SsoCdc6-1 from Sulfolobus solfataricus): binds ssDNA and dsDNA, has weak ATPase activity, undergoes autophosphorylation; Walker A mutant (K59A) abolishes ATPase and autophosphorylation; SsoCdc6-1 strongly inhibits ATPase and DNA helicase activity of S. solfataricus MCM—the first in vitro evidence of functional Cdc6-MCM interaction.","method":"Recombinant protein expression, EMSA, ATPase assay, autophosphorylation assay, Walker A mutagenesis, MCM helicase inhibition assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple in vitro biochemical assays with mutagenesis and reconstituted protein-protein functional interaction, single study","pmids":["12966100"],"is_preprint":false},{"year":2004,"finding":"Mitotic CDK Clb2/Cdc28 binds tightly to an N-terminal domain (NTD) of Cdc6 only when the NTD is phosphorylated on CDK consensus sites; Cdc6 in this complex cannot assemble pre-RCs; human CDKs with cyclins A, B, and E also bind phospho-NTD peptides; this Clb2-dependent mechanism contributes to preventing re-replication in vivo.","method":"In vitro binding assays with recombinant Clb2 and synthetic phospho-NTD peptides, co-IP, pre-RC assembly assay, in vivo re-replication assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted peptide binding with phosphorylation dependence, in vivo pre-RC assembly assay, and cross-species validation with human CDKs","pmids":["15496876"],"is_preprint":false},{"year":2004,"finding":"CDC6 is required for meiotic spindle formation in mouse oocytes; RNAi-mediated knockdown of CDC6 prevents meiotic spindle assembly without affecting resumption of meiosis, revealing a role for CDC6 in spindle organization beyond its established function in DNA replication.","method":"RNA interference knockdown of CDC6 in mouse oocytes, meiotic spindle immunofluorescence","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi with specific morphological phenotype, single lab","pmids":["15385409"],"is_preprint":false},{"year":2005,"finding":"S. cerevisiae Cdc6 binds cooperatively with ORC on origin DNA in an ATP-dependent manner, inducing a change in origin binding pattern that requires the Orc1 ATPase; single-particle EM reconstruction shows ORC-Cdc6 forms a ring-shaped complex with dimensions similar to the MCM helicase, predicted to contain six AAA+ subunits.","method":"ATP-dependent DNA binding assays, origin mutation analysis, single-particle electron microscopy reconstruction","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical reconstitution plus EM structural analysis, multiple origin mutations tested","pmids":["16228006"],"is_preprint":false},{"year":2005,"finding":"Recruitment of CDC6 (as a GAL4-DBD fusion) to a defined DNA array is sufficient to create a functional artificial origin of replication in mammalian cells; the ATPase domain of human Cdc6 is functionally important; N-terminal segments of ORC1/ORC2 are dispensable in this assay.","method":"GAL4-DBD fusion tethering assay, replication assay with geminin inhibition, ATPase domain mutant analysis","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional origin assay with domain mutants, single lab","pmids":["16322558"],"is_preprint":false},{"year":2005,"finding":"p53 activation by DNA damage enhances Cdc6 destruction via the APC; this destruction is triggered by inhibition of CDK2-mediated phosphorylation of CDC6 at serine 54; suppression of p53 stabilizes Cdc6, leading to more replicating cells—an effect reversed by reducing Cdc6 levels.","method":"DNA damage treatment, CDK2 inhibition, p53 knockdown, Cdc6 stability assays, phosphorylation-site mutagenesis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation-site mutagenesis with p53-pathway epistasis, single lab","pmids":["16055707"],"is_preprint":false},{"year":2006,"finding":"High levels of Cdc6 repress transcription of the INK4/ARF locus (p15INK4b, ARF, p16INK4a) through a replication origin (RD-INK4/ARF) that assembles Cdc6/Orc2/MCM complexes; Cdc6 overexpression recruits histone deacetylases and induces heterochromatinization of this locus; Cdc6 has cellular immortalization and transformation activities in cooperation with Ras.","method":"ChIP for Cdc6/Orc2/MCM at INK4/ARF locus, RNAi-induced heterochromatinization, HDAC recruitment assay, transformation assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP, HDAC recruitment, and functional transformation assays with multiple orthogonal methods in single study","pmids":["16572177"],"is_preprint":false},{"year":2006,"finding":"Cdc6 depletion during S phase (not G1) slows DNA replication and leads to mitotic lethality; Cdc6-depleted S-phase cells show fewer newly fired origins but active established replication forks; loss of Cdc6 in S phase fails to activate Chk1 kinase.","method":"RNAi-mediated depletion in synchronous G1 vs S-phase cells, origin firing analysis, Chk1 activation assay, mitotic outcome analysis","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi in synchronized cell populations with multiple functional readouts, single lab","pmids":["16439999"],"is_preprint":false},{"year":2006,"finding":"Caspase-3 cleaves Cdc6 at D290/S and D442/G sites during apoptosis; resulting truncated fragments (p32-tCdc6 and p49-tCdc6) promote apoptosis, perturb MCM2 (but not Orc2) chromatin loading, and activate ATM and ATR kinases with kinetics consistent with Chk1/2 phosphorylation.","method":"In vitro caspase-3 cleavage mapping, ectopic expression of tCdc6 fragments, chromatin loading assay for MCM2/Orc2, ATM/ATR kinase activation assay, siRNA for ATM/ATR","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical cleavage site mapping with functional ATM/ATR activation assay and MCM loading, single lab","pmids":["16801388"],"is_preprint":false},{"year":2007,"finding":"Cdc6 ATPase activity is activated by ORC, regulates ORC-Cdc6 complex stability, and is suppressed by origin DNA; specific origin DNA sequences (particularly the A element) down-regulate Cdc6 ATPase, resulting in stable ORC-Cdc6-DNA complex formation competent for MCM loading; on non-origin DNA, Cdc6 ATPase promotes Cdc6 dissociation.","method":"In vitro ATPase assay with ORC and various DNA sequences, ATPase mutants, ORC-Cdc6 complex stability assays on different DNAs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro ATPase and complex stability assays with multiple DNA substrates and mutants, mechanistic model","pmids":["17314092"],"is_preprint":false},{"year":2007,"finding":"Cdc6 stability after UV irradiation or MMS-induced DNA damage is regulated by the HECT-family ubiquitin E3 ligase Huwe1 (Mule/ARF-BP1); Cdc6 directly binds Huwe1; Huwe1 polyubiquitinates Cdc6 in vitro; this pathway is independent of p53, Cdc6 CDK-phosphorylation sites, and APC-Cdh1; it is conserved in yeast (Tom1 ortholog) and is associated with Cdc6 release from chromatin.","method":"Co-IP of Cdc6 with Huwe1, in vitro ubiquitination assay, Huwe1 knockdown, Tom1 deletion in yeast, UV/MMS treatment","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitination, co-IP, knockdown in human cells plus yeast ortholog validation; multiple orthogonal methods","pmids":["17567951"],"is_preprint":false},{"year":2011,"finding":"Cdc6 represses CDH1 (E-cadherin) transcription by binding to E-boxes in the CDH1 promoter, causing dissociation of the insulator CTCF, displacement of histone variant H2A.Z, and promoter heterochromatinization; mutational analysis identifies the Walker B motif and C-terminal region of Cdc6 as essential for this transcriptional suppression; CTCF displacement also activates adjacent replication origins.","method":"ChIP for Cdc6, CTCF, H2A.Z at CDH1 promoter, overexpression of Cdc6 in epithelial cells, mutational analysis of Walker B and C-terminal domain, replication origin activation assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP with mutagenesis and functional readouts (transcription and origin activation), multiple orthogonal methods","pmids":["22201124"],"is_preprint":false},{"year":2012,"finding":"Cryo-EM structure of S. cerevisiae ORC-Cdc6 on ARS1 origin DNA shows Cdc6 binding changes ORC conformation, particularly reorienting the Orc1 N-terminal BAH domain; a single Cdc6 extends the ORC footprint on origin DNA from both ends; the crescent-like ORC bends and wraps DNA.","method":"Single-particle cryo-EM, docking of archaeal Orc1/Cdc6 crystal structure, DNase I footprinting","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structural analysis with footprinting validation, single lab","pmids":["22405012"],"is_preprint":false},{"year":2012,"finding":"Cdc6 obstructs apoptosome assembly by forming stable complexes with cytochrome c-activated Apaf-1 monomers; this function depends on Cdc6's ATPase domain but not its cyclin-binding motif; in proliferating cells, Cdc6 suppresses seemingly unintended apoptosis while promoting cell proliferation.","method":"Co-immunoprecipitation of Cdc6 with Apaf-1, apoptosome assembly assay, ATPase domain mutant and cyclin-binding motif mutant analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with domain mutant analysis and functional apoptosome assay, single lab","pmids":["22493447"],"is_preprint":false},{"year":2012,"finding":"A yeast GSK-3 kinase homolog Mck1 promotes Cdc6 degradation by phosphorylating Cdc6 at Threonine-368 (a GSK-3 consensus site), leading to SCF(CDC4)-dependent proteolysis; mck1 deletion stabilizes Cdc6 in late S phase and mitosis; Mck1-dependent Cdc6 degradation is required to prevent DNA re-replication.","method":"Deletion analysis of MCK1, protein stability assays, phosphorylation-site mutagenesis (T368A), SCF genetic epistasis, DNA content analysis for re-replication","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation-site mutagenesis with genetic epistasis and re-replication readout, single lab","pmids":["23236290"],"is_preprint":false},{"year":2013,"finding":"During pre-RC assembly, ORC-Cdc6 forms an intermediate ORC-Cdc6-MCM2-7 (OCM) complex that is competent for MCM2-7 dimerization; the initial ORC-Cdc6-Cdt1-MCM2-7 (OCCM) complex is not competent for dimerization; MCM2-7 dimerization is a limiting, Cdc6-dependent step in pre-RC formation.","method":"Biochemical reconstitution of pre-RC assembly, MCM2-7 hexamer-interface mutants, complex analysis by EM, salt-sensitivity assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with defined mutants and EM validation, identification of a specific assembly intermediate, single lab","pmids":["24234446"],"is_preprint":false},{"year":2014,"finding":"A PIP-box in the N-terminus of Cdc6 mediates APC/C-CDH1-independent degradation of nuclear Cdc6 at the G1-S transition and during S phase via the CRL4-Cdt2 complex, preventing nuclear Cdc6 re-accumulation; Cdk1 contributes to nuclear export of Cdc6 at the S-to-G2 transition.","method":"PIP-box mutagenesis, Cdt2 knockdown, cell cycle synchronization, nuclear/cytoplasmic fractionation, Cdc6 stability assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PIP-box mutagenesis with Cdt2 depletion and fractionation, single lab","pmids":["24434580"],"is_preprint":false},{"year":2015,"finding":"Cdc6 ATPase activity is required for Cdc6 disengagement from the pre-RC after MCM helicase loading, not for MCM loading per se; an ATPase-defective Cdc6-E224Q mutant supports MCM loading but cells remain blocked in G1; degradation of Cdc6-E224Q after MCM loading restores apparently normal S phase, demonstrating that Cdc6 must disengage post-loading to allow helicase activation.","method":"Purified protein reconstitution of MCM loading with Cdc6 ATPase mutants, in vivo MCM chromatin association, conditional Cdc6 degradation experiments, S phase progression assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro loading with ATPase mutant plus in vivo conditional degradation rescue, mechanistically conclusive","pmids":["26305410"],"is_preprint":false},{"year":2016,"finding":"SCF(Cyclin F) ubiquitin ligase complex targets CDC6 for proteasomal degradation late in the cell cycle through defined sequence motifs; absence of Cyclin F or expression of a stable CDC6 mutant promotes DNA re-replication and genome instability in cells lacking Geminin.","method":"Co-IP of CDC6 with Cyclin F, ubiquitination assay, stable CDC6 mutant expression, re-replication assay by flow cytometry, Geminin depletion","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, ubiquitination assay, and functional re-replication assay with stable mutant, multiple orthogonal methods","pmids":["26818844"],"is_preprint":false},{"year":2016,"finding":"ORC1 represses Cyclin E gene (CCNE1) transcription by binding RB, the histone methyltransferase SUV39H1, and its repressive H3K9me3 mark; in contrast, CDC6 binds Cyclin E-CDK2 and removes RB from ORC1 in a feedback loop, thereby hyper-activating CCNE1 transcription; ORC1 and CDC6 thus have opposing effects on cell cycle commitment.","method":"Co-IP of ORC1 with RB and SUV39H1, ChIP for H3K9me3, promoter reporter assay, CDC6 overexpression removing RB from ORC1","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and ChIP with functional reporter assay, single lab","pmids":["27458800"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) at 3.9 Å shows Cdc6 winged-helix domain and positively charged loops contact origin DNA; flexible Mcm2-7 winged-helix domains engage ORC-Cdc6; DNA passes through both the ORC-Cdc6 and Mcm2-7 rings; Cdt1 embraces Mcm2, Mcm4, and Mcm6 comprising nearly half the hexamer.","method":"Cryo-EM structure determination at 3.9 Å resolution, crosslinking mass spectrometry","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic resolution cryo-EM structure of the complete loading intermediate with crosslinking MS validation","pmids":["28191893"],"is_preprint":false},{"year":2017,"finding":"Cdc6 is recruited to centrioles via Cyclin A and negatively regulates centrosome duplication by binding Sas-6, inhibiting stable Sas-6/STIL complex formation; Plk4 phosphorylates Cdc6 to disrupt Cdc6-Sas-6 interaction, counteracting Cdc6's inhibitory role on centrosome duplication; Cdc6 and Plk4 thus antagonistically control centrosome number.","method":"Co-IP of Cdc6 with Sas-6 and STIL, immunofluorescence for Cdc6 at centrosomes, Plk4 phosphorylation of Cdc6, centrosome duplication assay with Cdc6 mutants","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with kinase substrate identification and functional centrosome assay, single lab","pmids":["28447620"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM structures of Drosophila ORC with and without Cdc6 reveal that Orc1 and Orc4 constitute the primary DNA binding site; a Walker B loop of Orc1 contacts DNA, allosterically coupling DNA binding to the ATPase site; Cdc6 binding promotes DNA bending which facilitates MCM2-7 loading in vitro.","method":"Cryo-EM structure of Drosophila ORC ± Cdc6 on DNA, biochemical DNA-binding and MCM loading assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structural determination combined with functional biochemical assays, single study","pmids":["32848132"],"is_preprint":false},{"year":2020,"finding":"Two ORC-Cdc6-Cdt1-Mcm2-7 loading intermediates prior to DNA insertion were captured by cryo-EM: 'semi-attached OCCM' where Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 without the main body docking, and 'pre-insertion OCCM' where Mcm2-7 docks and origin DNA is bent adjacent to the Mcm2-Mcm5 open gate; molecular simulations show dynamic transition to the fully loaded state.","method":"Cryo-EM of loading intermediates (Mcm6-WHD truncation to slow reaction), molecular dynamics simulations","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structural intermediates with computational mechanistic analysis, multi-lab collaboration","pmids":["32669428"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of S. cerevisiae ORC-Cdc6 on ARS1 origin DNA at 3.3 Å reveals that Cdc6 contributes to origin DNA recognition via its winged-helix domain and initiator-specific motif; Cdc6 binding rearranges an α-helix in the Orc1 AAA+ domain and the Orc2 WHD, activating the Cdc6 ATPase and forming three Mcm2-7 recruitment sites absent in ORC alone.","method":"Cryo-EM at 3.3 Å resolution","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM revealing atomic-level mechanism of Cdc6-induced ORC activation","pmids":["34162887"],"is_preprint":false},{"year":2021,"finding":"Multiple short linear motifs (SLiMs) in the intrinsically disordered region (IDR) of CDC6 mediate cyclin-CDK-dependent and -independent interactions; the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interaction in G1; the CDC6 IDR regulates ORC1 self-interaction and controls ORC1 protein levels; Protein Phosphatase 1 binds ORC1 IDR causing de-phosphorylation at mitotic exit.","method":"Co-IP of CDC6 SLiM mutants with cyclins and ORC1, CDK2 kinase assays, cell cycle synchronization with protein level measurements","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SLiM mutagenesis with co-IP and kinase assays, single lab","pmids":["33761311"],"is_preprint":false},{"year":2024,"finding":"The deubiquitinase OTUD6A interacts with, deubiquitinates (removing K6-, K33-, and K48-linked polyubiquitin chains), and stabilizes CDC6 protein; OTUD6A promotes tumor cell proliferation and chemoresistance via CDC6 upregulation; OTUD6A-CDC6 axis is conserved in an in vivo bladder cancer model.","method":"Proteome-wide DUB screening, co-IP of OTUD6A with CDC6, in vitro deubiquitination assay, protein half-life assay, conditional Otud6a KO mouse model, xenograft model","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro deubiquitination reconstitution, co-IP, plus in vivo mouse KO and xenograft models; multiple orthogonal methods","pmids":["38685067"],"is_preprint":false}],"current_model":"CDC6 is an AAA+ ATPase that, upon binding to ORC on replication origins in G1, forms a ring-shaped ORC-Cdc6 complex that recruits Cdt1-bound Mcm2-7 hexamers (OCCM intermediate), loads the MCM2-7 double hexamer, and then disengages via its own ATPase activity to permit helicase activation; its abundance and localization are tightly controlled through cell cycle-regulated E2F-dependent transcription, Cyclin A/CDK2-mediated phosphorylation-driven nuclear export, and sequential ubiquitin-mediated proteolysis by APC/C-CDH1 (G1/quiescence), SCF-Cdc4 (G1/S), SCF-Cyclin F (late cell cycle), and Huwe1 (DNA damage), while its stabilization by the deubiquitinase OTUD6A can promote oncogenesis; beyond DNA replication licensing, CDC6 inhibits mitotic CDKs to couple S phase completion to mitotic entry, represses tumor suppressor loci (INK4/ARF, CDH1) through chromatin remodeling, inhibits apoptosome assembly via Apaf-1 binding, and is cleaved by caspase-3 during apoptosis to generate pro-apoptotic nuclear fragments."},"narrative":{"mechanistic_narrative":"CDC6 is an essential AAA+ ATPase that licenses eukaryotic DNA replication origins by cooperating with ORC to load the MCM2-7 replicative helicase, and it has been conserved from archaea and yeast to humans as a master regulator coupling origin firing to cell cycle progression [PMID:2656692, PMID:7641697, PMID:16228006]. Cdc6 binds ORC cooperatively on origin DNA in an ATP-dependent manner to form a ring-shaped, six-AAA+-subunit ORC-Cdc6 complex that bends origin DNA, rearranges ORC (including the Orc1 AAA+ domain and BAH/winged-helix elements), and creates the MCM2-7 recruitment sites absent from ORC alone [PMID:16228006, PMID:22405012, PMID:32848132, PMID:34162887]. Cdc6's own winged-helix and initiator-specific motifs contact origin DNA within the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) loading intermediate, through which DNA is threaded; the reaction proceeds via defined semi-attached and pre-insertion intermediates and an ORC-Cdc6-MCM (OCM) state competent for MCM2-7 dimerization [PMID:24234446, PMID:28191893, PMID:32669428]. Cdc6 ATPase activity, activated by ORC and suppressed by origin DNA, is required not for loading itself but for Cdc6 disengagement from the pre-RC after helicase loading, a step essential for subsequent helicase activation [PMID:17314092, PMID:26305410]. Cdc6 abundance, localization, and activity are tightly cell-cycle-controlled: human CDC6 transcription is driven by E2F factors and CDC6 cooperates with cyclin E to drive S-phase entry [PMID:9520412, PMID:9774682, PMID:11805305], while Cyclin A/CDK2 phosphorylation of N-terminal CDK sites drives nuclear-to-cytoplasmic relocalization and destruction of non-chromatin-bound Cdc6 to prevent re-replication [PMID:9889196, PMID:10806104]. Cdc6 is degraded by sequential ubiquitin pathways—APC/C-CDH1 in G1 and quiescence, SCF-Cdc4, CRL4-Cdt2, SCF-Cyclin F late in the cycle, and the HECT ligase Huwe1 after DNA damage—and CDK/Clb binding to phosphorylated Cdc6 also inhibits pre-RC assembly [PMID:10995389, PMID:15496876, PMID:17567951, PMID:24434580, PMID:26818844]. Beyond licensing, Cdc6 restrains mitotic entry by binding and inhibiting CDK complexes and acting cooperatively with Sic1 [PMID:1600944, PMID:11460169, PMID:12554670], represses the INK4/ARF and CDH1 loci through origin-coupled chromatin remodeling and HDAC recruitment with transforming activity [PMID:16572177, PMID:22201124], and obstructs apoptosome assembly by sequestering Apaf-1, while caspase-3 cleavage of Cdc6 during apoptosis generates nuclear pro-apoptotic, replication-inhibitory fragments [PMID:22493447, PMID:12151338, PMID:14517333]. The deubiquitinase OTUD6A stabilizes CDC6 to promote tumor proliferation and chemoresistance [PMID:38685067].","teleology":[{"year":1989,"claim":"Established CDC6 as an essential, conserved nucleotide-binding protein required for S-phase entry, defining the gene's core role before any mechanism was known.","evidence":"Complementation cloning, gene disruption, and sequencing in S. cerevisiae","pmids":["2656692"],"confidence":"Medium","gaps":["No biochemical activity demonstrated","Molecular role at origins unknown"]},{"year":1990,"claim":"Showed CDC6 expression is cell-cycle periodic, peaking at G1/S, framing it as a transcriptionally gated licensing factor.","evidence":"Synchronized yeast cultures, Northern blotting, promoter analysis","pmids":["2246267"],"confidence":"Medium","gaps":["Transcription factors driving periodicity not yet defined","Protein-level regulation unaddressed"]},{"year":1995,"claim":"Demonstrated that Cdc6 is required for replication initiation and that its absence uncouples replication from mitosis, revealing a checkpoint-like coupling function.","evidence":"Yeast genetics, synchronization, FISH, transcription factor mutants","pmids":["7641697"],"confidence":"High","gaps":["Molecular mechanism of mitotic coupling unresolved","Direct origin role not yet shown"]},{"year":1996,"claim":"Identified Cdc6 as forming origin pre-replicative complexes distinct from ORC and as a binding partner/inhibitor of B-type cyclin/Cdc28 kinase, linking origin licensing to CDK control.","evidence":"Genomic footprinting at yeast origins; co-IP, pulldown, in vitro kinase assays, deletion mutants","pmids":["8978693","8930895"],"confidence":"High","gaps":["Mechanism of CDK inhibition not structurally defined","How Cdc6 nucleates MCM loading unknown"]},{"year":1998,"claim":"Extended the model to humans: E2F-driven transcription, ORC1 and cyclin-CDK association, NLS-independent nuclear localization, and antibody-blocked replication established CDC6 as a conserved human licensing factor.","evidence":"Promoter-reporter, in vivo footprinting, antibody microinjection, fractionation, co-IP, NLS mutagenesis in human cells","pmids":["9520412","9566895","9774682"],"confidence":"High","gaps":["Enzymatic requirement not yet tested in human protein","Mechanism of S-phase nuclear elimination unresolved"]},{"year":1999,"claim":"Defined Cdc6 ATPase activity as essential for replication and showed Cyclin A/CDK2 phosphorylation controls its localization, establishing both the catalytic requirement and the re-replication safeguard.","evidence":"Recombinant ATP binding/hydrolysis with Walker A/B mutants and microinjection; in vitro kinase, domain mapping, fractionation, immunofluorescence","pmids":["10436018","9889196","9857179"],"confidence":"High","gaps":["Step in licensing requiring ATPase not yet pinpointed","How phosphorylation triggers export mechanistically unclear"]},{"year":2000,"claim":"Resolved that ubiquitin-mediated proteolysis (APC/C-CDH1 in G1/quiescence; SCF-Cdc4 in yeast) and Cyclin A-CDK2 phosphorylation selectively destroy non-chromatin-bound Cdc6, preventing re-licensing.","evidence":"In vitro APC/CDH1 ubiquitination, depletion, D-box/KEN-box mutagenesis; chromatin fractionation with recombinant Cyclin A-CDK2; yeast cdc4 epistasis","pmids":["10995389","10806104","10085159","9442103"],"confidence":"High","gaps":["Coordination between distinct degradation pathways unresolved","Chromatin-bound vs soluble pool discrimination mechanism incomplete"]},{"year":2000,"claim":"Crystallography of an archaeal Cdc6 ortholog revealed the AAA+ fold with a DNA-binding winged-helix domain, providing the structural framework for origin recognition and showing both domains are functionally required.","evidence":"X-ray crystallography (2.0 A), WH and ATPase mutagenesis with S. pombe functional assays","pmids":["11030343"],"confidence":"High","gaps":["Eukaryotic complex architecture not yet visualized","How WH domain engages origin DNA in ORC context unknown"]},{"year":2001,"claim":"Showed Cdc6 directly inactivates mitotic CDKs (with Sic1) and recruits Cyclin E-CDK2 to chromatin via RXL motifs, formalizing dual roles in licensing and mitotic restraint.","evidence":"Genetic CDK-domain deletion and epistasis, co-IP, in vitro CDK assays; Xenopus extract chromatin recruitment with RXL/MRAIL mutagenesis","pmids":["11460169","11257126"],"confidence":"High","gaps":["Quantitative contribution of Cdc6 vs Sic1 to mitotic exit unclear","How chromatin-localized CDK2 acts on licensing not fully defined"]},{"year":2002,"claim":"Demonstrated Cdc6 alone suffices to load MCM and license origins in quiescent cells in an ATPase-dependent, cyclin E-CDK2-synergistic manner, and is required to re-establish replication competence in meiotic oocytes.","evidence":"Adenoviral wild-type/mutant Cdc6 with MCM chromatin assays; Xenopus oocyte Cdc6 protein injection with synthesis inhibition","pmids":["11805305","12384699"],"confidence":"High","gaps":["Stoichiometry of MCM loading not defined","Mechanism of CDK isoform specificity unresolved"]},{"year":2003,"claim":"Provided first reconstituted Cdc6-MCM functional interaction (archaeal) and showed human Cdc6 overexpression in G2 blocks mitosis via a Chk1-dependent checkpoint, reinforcing replication-mitosis coupling.","evidence":"Archaeal recombinant biochemistry (EMSA, ATPase, MCM inhibition, Walker A mutant); G2 overexpression with UCN-01 and Cdc25 epistasis","pmids":["12966100","12554670"],"confidence":"Medium","gaps":["Physiological relevance of G2 checkpoint role unclear","Direct human Cdc6-MCM contacts not yet structurally defined"]},{"year":2005,"claim":"Visualized the ring-shaped ORC-Cdc6 complex and showed Cdc6 tethering creates an artificial mammalian origin, establishing Cdc6 as the architectural trigger for origin assembly.","evidence":"ATP-dependent DNA binding, single-particle EM; GAL4-DBD tethering replication assay with ATPase mutants","pmids":["16228006","16322558"],"confidence":"High","gaps":["Atomic detail of DNA engagement not yet resolved","MCM recruitment geometry unknown at this resolution"]},{"year":2006,"claim":"Revealed a non-licensing oncogenic function: high Cdc6 represses the INK4/ARF tumor suppressor locus via origin-coupled HDAC recruitment and heterochromatinization, with Ras-cooperative transforming activity.","evidence":"ChIP for Cdc6/Orc2/MCM at INK4/ARF, HDAC recruitment, RNAi heterochromatinization, transformation assays","pmids":["16572177"],"confidence":"High","gaps":["Generality across other loci not yet established","Link between origin function and repression mechanistically incomplete"]},{"year":2007,"claim":"Defined the regulatory logic of Cdc6 ATPase—activated by ORC, suppressed by origin DNA—to stabilize productive ORC-Cdc6-DNA complexes at origins while dissociating Cdc6 from non-origin DNA.","evidence":"In vitro ATPase and complex-stability assays with multiple DNA substrates and mutants; co-IP, in vitro ubiquitination and Huwe1 knockdown for damage-induced turnover","pmids":["17314092","17567951"],"confidence":"High","gaps":["Coupling of ATPase state to MCM loading step not yet resolved","Damage-induced chromatin release mechanism partly defined"]},{"year":2011,"claim":"Showed Cdc6 represses CDH1 (E-cadherin) by E-box binding, CTCF dissociation, and H2A.Z displacement, extending its chromatin-repressive and origin-activating activities to an EMT-relevant locus.","evidence":"ChIP for Cdc6/CTCF/H2A.Z, Walker B and C-terminal mutagenesis, origin activation assay","pmids":["22201124"],"confidence":"High","gaps":["In vivo tumor relevance not directly tested here","How replication and repression activities are coordinated unclear"]},{"year":2012,"claim":"Provided cryo-EM of ORC-Cdc6 on origin DNA and revealed Cdc6's anti-apoptotic function via Apaf-1 sequestration, broadening its role to cell survival.","evidence":"Single-particle cryo-EM with footprinting; co-IP with Apaf-1, apoptosome assembly assay with domain mutants","pmids":["22405012","22493447"],"confidence":"Medium","gaps":["Apaf-1 interaction structurally undefined","Physiological threshold of anti-apoptotic activity unknown"]},{"year":2013,"claim":"Identified the ORC-Cdc6-MCM (OCM) intermediate as the Cdc6-dependent, dimerization-competent step distinguishing it from the earlier OCCM, defining the rate-limiting transition in helicase loading.","evidence":"Biochemical pre-RC reconstitution, MCM interface mutants, EM, salt-sensitivity assays","pmids":["24234446"],"confidence":"High","gaps":["Conformational basis of dimerization competence not yet atomic","Timing relative to Cdt1 release unclear"]},{"year":2015,"claim":"Demonstrated definitively that Cdc6 ATPase is required for post-loading disengagement of Cdc6, not MCM loading itself—resolving a long-standing question about the catalytic step.","evidence":"Reconstituted loading with Cdc6-E224Q ATPase mutant plus in vivo conditional degradation rescue","pmids":["26305410"],"confidence":"High","gaps":["How disengagement enables helicase activation mechanistically unclear","Structural state of disengaged complex undefined"]},{"year":2016,"claim":"Added SCF-Cyclin F as a late-cycle CDC6 degradation pathway guarding against re-replication, and showed ORC1/CDC6 form an opposing transcriptional circuit at CCNE1, integrating licensing with cell-cycle commitment.","evidence":"Co-IP, ubiquitination, re-replication flow cytometry with stable mutant; ORC1-RB-SUV39H1 co-IP/ChIP with CDC6 RB-displacement assay","pmids":["26818844","27458800"],"confidence":"High","gaps":["Redundancy among degradation pathways not quantified","Direct demonstration of feedback circuit in normal proliferation incomplete"]},{"year":2017,"claim":"Near-atomic cryo-EM of the OCCM and discovery of Cdc6 at centrioles revealed both the structural basis of origin licensing and a moonlighting role limiting centrosome duplication.","evidence":"Cryo-EM of OCCM at 3.9 A with crosslinking MS; co-IP with Sas-6/STIL, centrosome assays, Plk4 phosphorylation","pmids":["28191893","28447620"],"confidence":"High","gaps":["DNA insertion step not captured","Physiological extent of centrosome regulation unclear"]},{"year":2020,"claim":"Captured loading intermediates and DNA bending mechanics by cryo-EM and MD, showing how Cdc6 binding promotes DNA bending and stepwise MCM2-7 engagement toward the loaded state.","evidence":"Cryo-EM of Drosophila ORC +/- Cdc6 and yeast semi-attached/pre-insertion OCCM with molecular dynamics","pmids":["32848132","32669428"],"confidence":"High","gaps":["Full DNA insertion and gate closure dynamics still inferred","How ATPase activity drives these transitions not resolved"]},{"year":2021,"claim":"Resolved at 3.3 A how Cdc6 contributes to origin recognition and allosterically activates its own ATPase by rearranging ORC, and defined CDC6 disordered-region SLiMs governing cyclin-CDK and ORC1 interactions.","evidence":"Cryo-EM at 3.3 A; SLiM mutagenesis with co-IP and CDK2 assays","pmids":["34162887","33761311"],"confidence":"High","gaps":["Dynamics of ATPase activation during loading still modeled, not observed","Functional hierarchy of individual SLiMs incomplete"]},{"year":2024,"claim":"Identified OTUD6A as a deubiquitinase that stabilizes CDC6 to drive tumor proliferation and chemoresistance, providing a disease-relevant post-translational control point.","evidence":"DUB screen, co-IP, in vitro deubiquitination, half-life assays, conditional KO mouse, xenograft bladder cancer model","pmids":["38685067"],"confidence":"High","gaps":["Whether OTUD6A acts on chromatin-bound vs soluble CDC6 unknown","Generality across cancer types not established"]},{"year":null,"claim":"How Cdc6 ATPase-driven disengagement is mechanically coupled to helicase activation, and how its licensing function is integrated with its chromatin-repressive, anti-apoptotic, and centrosomal roles in a single cell, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of the disengaged post-loading state","Mechanism connecting origin function to transcriptional repression unclear","How moonlighting functions are temporally partitioned from licensing unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[3,10,23,37,45]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[3,10,37]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[18,28,48,52]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,21,29,41]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[34,39,47]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,12,16,26]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[5,14,34]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12,16]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[49]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[4,11,23,43,45,48]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,21,27,12]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[34,39]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[25,26,41]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[15,38,46,54]}],"complexes":["ORC-Cdc6","ORC-Cdc6-Cdt1-Mcm2-7 (OCCM)","ORC-Cdc6-Mcm2-7 (OCM)","pre-replicative complex"],"partners":["ORC1","MCM2-7","CDT1","CCNA2","CCNE1","HUWE1","OTUD6A","APAF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99741","full_name":"Cell division control protein 6 homolog","aliases":["CDC6-related protein","Cdc18-related protein","HsCdc18","p62(cdc6)","HsCDC6"],"length_aa":560,"mass_kda":62.7,"function":"Involved in the initiation of DNA replication. Also participates in checkpoint controls that ensure DNA replication is completed before mitosis is initiated","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q99741/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CDC6","classification":"Common Essential","n_dependent_lines":1206,"n_total_lines":1208,"dependency_fraction":0.9983443708609272},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CDC6","total_profiled":1310},"omim":[{"mim_id":"621044","title":"RING FINGER PROTEIN 157; RNF157","url":"https://www.omim.org/entry/621044"},{"mim_id":"616485","title":"ZINC FINGER- AND BTB DOMAIN-CONTAINING PROTEIN 21; ZBTB21","url":"https://www.omim.org/entry/616485"},{"mim_id":"615929","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 17; ANKRD17","url":"https://www.omim.org/entry/615929"},{"mim_id":"615167","title":"LEUCINE-RICH REPEATS- AND WD REPEAT DOMAIN-CONTAINING PROTEIN 1; LRWD1","url":"https://www.omim.org/entry/615167"},{"mim_id":"614938","title":"VAULT RNA 2-1; VTRNA2-1","url":"https://www.omim.org/entry/614938"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":25.7},{"tissue":"lymphoid 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cell","url":"https://pubmed.ncbi.nlm.nih.gov/14517333","citation_count":26,"is_preprint":false},{"pmid":"31100539","id":"PMC_31100539","title":"Downregulation of Cdc6 inhibits tumorigenesis of osteosarcoma in vivo and in vitro.","date":"2019","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/31100539","citation_count":25,"is_preprint":false},{"pmid":"30450027","id":"PMC_30450027","title":"CDC6 mRNA Expression Is Associated with the Aggressiveness of Prostate Cancer.","date":"2018","source":"Journal of Korean medical science","url":"https://pubmed.ncbi.nlm.nih.gov/30450027","citation_count":25,"is_preprint":false},{"pmid":"23236290","id":"PMC_23236290","title":"A yeast GSK-3 kinase Mck1 promotes Cdc6 degradation to inhibit DNA re-replication.","date":"2012","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23236290","citation_count":25,"is_preprint":false},{"pmid":"36257938","id":"PMC_36257938","title":"LCAT1 is an oncogenic LncRNA by stabilizing the IGF2BP2-CDC6 axis.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36257938","citation_count":24,"is_preprint":false},{"pmid":"22493447","id":"PMC_22493447","title":"Cdc6 protein obstructs apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22493447","citation_count":24,"is_preprint":false},{"pmid":"12628342","id":"PMC_12628342","title":"Down to the origin: Cdc6 protein and the competence to replicate.","date":"2003","source":"Trends in cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/12628342","citation_count":23,"is_preprint":false},{"pmid":"17690116","id":"PMC_17690116","title":"Cdc18/CDC6 activates the Rad3-dependent checkpoint in the fission yeast.","date":"2007","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/17690116","citation_count":23,"is_preprint":false},{"pmid":"28985365","id":"PMC_28985365","title":"Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype.","date":"2017","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28985365","citation_count":22,"is_preprint":false},{"pmid":"8552037","id":"PMC_8552037","title":"The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination.","date":"1995","source":"Molecular & general genetics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/8552037","citation_count":21,"is_preprint":false},{"pmid":"38685067","id":"PMC_38685067","title":"Deubiquitination of CDC6 by OTUD6A promotes tumour progression and chemoresistance.","date":"2024","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/38685067","citation_count":20,"is_preprint":false},{"pmid":"33020506","id":"PMC_33020506","title":"Cdc6 disruption leads to centrosome abnormalities and chromosome instability in pancreatic cancer cells.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33020506","citation_count":20,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":45336,"output_tokens":13613,"usd":0.170101,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":25447,"output_tokens":7477,"usd":0.15708,"stage2_stop_reason":"end_turn"},"total_usd":0.327181,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1989,\n      \"finding\": \"CDC6 gene encodes an essential protein for S phase entry in S. cerevisiae; the deduced protein sequence contains a conserved nucleotide-binding site, and disruption of CDC6 is lethal for mitotic growth.\",\n      \"method\": \"Complementation cloning, gene disruption, DNA sequencing\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation and gene disruption with defined lethal phenotype, single study\",\n      \"pmids\": [\"2656692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"CDC6 mRNA is periodically expressed in the yeast cell cycle, peaking at the G1/S boundary, and the CDC6 promoter contains sequence elements (similar to those in other cell cycle-regulated genes) that drive this periodic transcription.\",\n      \"method\": \"Synchronized culture experiments (alpha-factor arrest, elutriation), Northern blotting, promoter sequence analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent synchronization methods, single lab\",\n      \"pmids\": [\"2246267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Constitutive CDC6 expression in yeast delays initiation of M phase in a manner dependent on the Wee1/Mik1 mitotic inhibitor kinases and is counteracted by Cdc25/MIH1 phosphatases, indicating CDC6 indirectly inhibits activation of p34cdc2/CDC28 M-phase kinase; CDC6 thus has a dual role in requiring DNA replication initiation and suppressing nuclear division.\",\n      \"method\": \"Constitutive expression of CDC6 in budding and fission yeast, genetic interaction analysis with wee1, mik1, cdc25, MIH1 mutants\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in two yeast species, single lab\",\n      \"pmids\": [\"1600944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Purified S. cerevisiae Cdc6 protein binds rATP and rGTP upon UV cross-linking and catalyzes DNA-independent hydrolysis of purine nucleoside triphosphates, consistent with an ATPase/GTPase activity that may regulate replication initiation.\",\n      \"method\": \"Recombinant protein expression in E. coli, UV cross-linking nucleotide binding assay, ATPase/GTPase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assay with purified protein, single lab, no mutagenesis reported\",\n      \"pmids\": [\"8083240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Cdc6 is an unstable protein whose de novo synthesis in G1 (driven first by Swi5, then by MBF/SBF transcription factors) is required for initiation of DNA replication; cells lacking Cdc6 fail to replicate DNA but still undergo mitosis ('reductional anaphase'), demonstrating that Cdc6 deficiency uncouples DNA replication from mitotic entry.\",\n      \"method\": \"Yeast genetics, cell synchronization, fluorescence in situ hybridization (FISH), transcription factor mutant analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic approaches and FISH in budding yeast, replicated across conditions\",\n      \"pmids\": [\"7641697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"ORC and Cdc6 form distinct chromatin complexes at replication origins in S. cerevisiae: origins oscillate between an ORC-dependent post-replicative state and a Cdc6-dependent pre-replicative state during the cell cycle.\",\n      \"method\": \"Genomic footprinting at single-copy chromosomal origins in yeast\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct in vivo footprinting at endogenous loci, multiple origins tested\",\n      \"pmids\": [\"8978693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Yeast Cdc6 physically interacts with B-type cyclin/Cdc28 kinase complexes (not Cln/Cdc28); Cdc6 is a substrate and inhibitor of Cdc28 kinase in vitro; the Cdc28-interaction domain of Cdc6 is required for its essential growth function and for restraining mitosis.\",\n      \"method\": \"Co-immunoprecipitation, p13-agarose pulldown, affinity chromatography with bacterially produced Cdc6, in vitro kinase assay, deletion mutant analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reciprocal pulldown, in vitro kinase assay, and genetic complementation with deletion mutant, single lab\",\n      \"pmids\": [\"8930895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human CDC6 transcription is regulated by E2F transcription factors; E2F binding sites in the CDC6 promoter are required for cell cycle-regulated expression; microinjection of anti-CDC6 antibody blocks initiation of DNA replication in human tumor cells.\",\n      \"method\": \"Promoter-reporter assay, in vivo footprinting, microinjection of antibody, E2F overexpression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (promoter analysis, in vivo footprinting, antibody microinjection), replicated by independent labs\",\n      \"pmids\": [\"9520412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human CDC6/Cdc18 is nuclear in G1 and is selectively eliminated from the nucleus at the onset of S phase; it associates with human Orc1 protein and cyclin-CDK complexes; nuclear localization is independent of its nuclear localization signal, implying association with other nuclear proteins.\",\n      \"method\": \"Epitope-tagged protein cell cycle fractionation, co-immunoprecipitation with Orc1 and cyclin-CDKs, site-directed mutagenesis of NLS\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, subcellular fractionation, NLS mutagenesis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"9566895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Mammalian CDC6 promoter is activated by E2F proteins; E2F binding sites are required for serum-stimulated and cell cycle-regulated expression; CDC6 cooperates with cyclin E to induce S phase entry; microinjection of anti-CDC6 antiserum blocks DNA synthesis.\",\n      \"method\": \"Promoter-reporter assay, E2F overexpression, co-transfection with cyclin E, antibody microinjection\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, consistent with independent reports\",\n      \"pmids\": [\"9774682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Recombinant human Cdc6 (HsCdc6) specifically binds ATP and slowly hydrolyzes it; Walker A and B motif mutants are defective in ATP binding/hydrolysis and display aberrant conformations in the presence of nucleotides; microinjection of either mutant inhibits DNA replication in G1 cells, demonstrating that Cdc6 ATPase activity is essential for DNA replication.\",\n      \"method\": \"Recombinant protein expression, ATP binding and hydrolysis assays, mutagenesis of Walker A and B motifs, microinjection into human cells\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis plus in vivo functional validation, single lab\",\n      \"pmids\": [\"10436018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Cdc6 protein causes premature entry into S phase: addition of recombinant Cdc6 to permeabilized G1 nuclei induces up to 82% of nuclei to initiate DNA replication and accelerates G1 progression in a mammalian cell-free system; quiescent cells lack Cdc6 and fail to load MCM proteins onto chromatin.\",\n      \"method\": \"Mammalian cell-free DNA replication system, recombinant Cdc6 addition, immunoblot for Cdc6 and MCM chromatin association\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution in cell-free system with quantitative readout, single lab\",\n      \"pmids\": [\"9857179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Mammalian CDC6 is phosphorylated specifically by Cyclin A/CDK2 (not Cyclin E or Cyclin B) via an N-terminal Cy-motif; phosphorylation of three N-terminal CDK consensus sites regulates CDC6 subcellular localization; CDC6 is nuclear in G1 and relocalizes to the cytoplasm upon Cyclin A/CDK2 activation, suggesting phosphorylation prevents re-replication.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, cyclin binding domain mapping, ectopic Cyclin A/E expression, subcellular fractionation and immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay plus in vivo co-IP, domain mapping, and localization experiments; multiple orthogonal methods\",\n      \"pmids\": [\"9889196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"S. cerevisiae Cdc6 protein is ubiquitinated in vivo and degraded by a Cdc4-dependent (SCF) ubiquitin-mediated proteolytic pathway at the late G1/early S phase transition.\",\n      \"method\": \"In vivo ubiquitination assay, analysis of Cdc6 stability in cdc4 mutants, cell cycle synchronization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo ubiquitination detection and genetic epistasis with cdc4, single lab\",\n      \"pmids\": [\"10085159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"After formation of pre-initiation complexes (ORC, Cdc6, MCM on chromatin), Cdc6 is rapidly removed from chromatin in Xenopus extracts, possibly via a cdk2-activated ubiquitin-dependent proteolytic pathway; inhibition of this removal blocks DNA replication initiation; subsequent initiation steps are independent of ORC and Cdc6 but dependent on cdk2 activity.\",\n      \"method\": \"Xenopus laevis egg extract system, chromatin fractionation, cdk2 inhibition, ubiquitin pathway interference\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted cell-free system with defined biochemical perturbations, multiple epistasis tests in single study\",\n      \"pmids\": [\"9442103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Human CDC6 is targeted for ubiquitin-mediated proteolysis by APC/C-CDH1 in G1 and quiescent cells; point mutations in both the destruction box and KEN-box motifs together stabilize CDC6; APC/CDH1 ubiquitinates CDC6 in vitro; APC and CDH1 are required and limiting for CDC6 proteolysis in vivo.\",\n      \"method\": \"In vitro ubiquitination assay with purified APC/CDH1, APC/CDH1 depletion, mutagenesis of destruction box and KEN box, stability assays in G1 and quiescent cells\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution of ubiquitination plus in vivo depletion with mutagenesis; multiple orthogonal methods\",\n      \"pmids\": [\"10995389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a process requiring nuclear import and phosphorylation by a chromatin-bound kinase; recombinant Cyclin A-CDK2 completely substitutes for the nucleus in promoting destruction of soluble Cdc6, suggesting that Cyclin A-CDK2 phosphorylation destroys free Cdc6 not assembled into replication complexes.\",\n      \"method\": \"Mammalian cell extract in vitro replication system, chromatin fractionation, recombinant Cyclin A-CDK2 addition, nuclear import inhibition\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cell-free biochemical reconstitution with defined kinase substitution, single lab\",\n      \"pmids\": [\"10806104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cdc6 stability in S. cerevisiae is regulated by Cdc28 (CDK1)/Clb kinase activity; Cdc6 mutants lacking Cdc28 phosphorylation sites are stabilized; loss of Cdc28/Clb kinase activity allows accumulation of Cdc6 protein in mitotic-arrested cells.\",\n      \"method\": \"Cell cycle synchronization, protein stability assays, phosphorylation-site mutant analysis, temperature-sensitive cdc28 mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation-site mutagenesis and genetic epistasis, single lab\",\n      \"pmids\": [\"10734126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of an archaeal Cdc6 ortholog (2.0 Å) reveals an AAA+-type nucleotide binding fold bound to Mg·ADP and a winged-helix (WH) domain similar to known DNA-binding modules; mutagenesis of the WH domain of S. pombe Cdc18 shows this region is required for function in vivo; nucleotide binding/hydrolysis by Cdc6/Cdc18 is required for S phase progression and for maintenance of S-phase checkpoint control.\",\n      \"method\": \"X-ray crystallography (2.0 Å), site-directed mutagenesis of WH domain and ATPase motifs, in vivo functional assays in S. pombe\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure combined with mutagenesis and in vivo functional validation\",\n      \"pmids\": [\"11030343\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cyclin E binds the N-terminal region of Cdc6 via RXL (Cy) motifs on Cdc6 and the substrate-selection (MRAIL) motif on Cyclin E; this interaction localizes Cyclin E-CDK2 to chromatin in an ORC- and Cdc6-dependent manner; mutation of the RXL motifs in Cdc6 abrogates Cyclin E binding and rescuing of replication in Cdc6-depleted Xenopus extracts.\",\n      \"method\": \"Xenopus egg extract chromatin assembly assay, domain mapping, site-directed mutagenesis of RXL and MRAIL motifs, rescue assays with Cdc6-depleted extracts\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis of both interacting proteins and rescue experiments\",\n      \"pmids\": [\"11257126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cdc6 expression in fission yeast G2 cells overrides controls that ensure one S phase per cycle by re-firing replication origins and recruiting MCM Cdc21 to chromatin; co-expression of Cdt1 greatly amplifies this re-replication; Cdt1 may stabilize Cdc18 on chromatin.\",\n      \"method\": \"Inducible expression in S. pombe G2 cells, FACS analysis, MCM chromatin association, phosphorylation-site mutant analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic inducible system with chromatin association readout, single lab\",\n      \"pmids\": [\"11532929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cdc6 cooperates with Sic1 to inactivate mitotic CDKs during late mitosis in S. cerevisiae; deletion of the CDK-interacting domain of Cdc6 causes a mitotic exit delay that is accentuated in the absence of Sic1 or cyclin degradation; Cdc6, like Sic1, binds CDK complexes in vivo and downregulates them in vitro.\",\n      \"method\": \"Genetic deletion of Cdc6 CDK-binding domain, double mutant analysis with sic1 and cyclin degradation mutants, co-immunoprecipitation, in vitro CDK kinase assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro CDK inhibition, in vivo co-IP, and epistasis with multiple genetic backgrounds, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"11460169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human Cdc6 is rapidly destroyed by a proteasome- and ubiquitin-dependent pathway during early apoptosis induced by adozelesin (p53-independent) and by a separate caspase-dependent pathway during TNF-α-induced apoptosis; the proteasome-dependent pathway is conserved in S. cerevisiae.\",\n      \"method\": \"Western blot of Cdc6 during apoptosis, proteasome inhibitor, p53-null cell lines, cross-species comparison\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological pathway dissection in human and yeast cells, single lab\",\n      \"pmids\": [\"12006651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Mammalian Cdc6 expression alone (via adenoviral vector) is sufficient to induce stable MCM chromatin association in quiescent cells with low CDK activity; Cdc6 ATP-binding site mutation severely impairs MCM loading; Cdc6 synergizes with cyclin E/CDK2 (but not cyclin A/CDK2) to induce semiconservative DNA replication in quiescent cells.\",\n      \"method\": \"Adenoviral expression of wild-type and mutant Cdc6 in quiescent cells, chromatin fractionation for MCM, semiconservative replication assay, CDK co-expression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional dissection with ATPase mutants, CDK specificity determination, and MCM loading assay in intact mammalian cells\",\n      \"pmids\": [\"11805305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Xenopus Cdc6 synthesis during meiosis I (at germinal vesicle breakdown, GVBD) is necessary and sufficient for re-establishing DNA replication competence in oocytes; injection of Cdc6 protein into GVBD oocytes induces DNA replication in the absence of other protein synthesis.\",\n      \"method\": \"Xenopus oocyte injection of recombinant Cdc6 protein, replication competence assay, protein synthesis inhibition\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution by direct protein injection with protein synthesis inhibitor control, single lab\",\n      \"pmids\": [\"12384699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human Cdc6 is specifically cleaved by caspase-3 during apoptosis; expression of a caspase-uncleavable Cdc6 mutant attenuates apoptosis, demonstrating that Cdc6 cleavage facilitates cell death and prevents a wounded cell from replicating.\",\n      \"method\": \"Western blot of Cdc6 cleavage during apoptosis in multiple cell lines, expression of cleavage-resistant Cdc6 mutant, apoptosis assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — caspase-3 cleavage site identification with dominant-negative mutant, single lab\",\n      \"pmids\": [\"12151338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Caspase-3 cleaves Cdc6 at SEVD442/G site during apoptosis, generating p49-tCdc6 that lacks the C-terminal nuclear export sequence; p49-tCdc6 is retained in the nucleus (resistant to Cyclin A-CDK2-mediated export), acts as a dominant negative inhibitor of DNA replication, and promotes apoptosis when ectopically expressed.\",\n      \"method\": \"In vitro caspase-3 cleavage assay, mutagenesis of cleavage sites, subcellular fractionation, ectopic expression of truncated Cdc6, apoptosis assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical cleavage site mapping with functional nuclear retention and apoptosis assays, single lab\",\n      \"pmids\": [\"14517333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human Cdc6 overexpression in G2 phase prevents entry into mitosis via a Chk1-dependent checkpoint; this block is abolished by constitutively active Cyclin B/CDK1, Cdc25B, or Cdc25C, or by the Chk1 inhibitor UCN-01; overexpressed Cdc6 in G2 induces Chk1 phosphorylation.\",\n      \"method\": \"Ectopic HuCdc6 overexpression in G2-phase cells, kinase inhibitor (UCN-01), Cdc25 co-expression, flow cytometry, Chk1 phosphorylation assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological epistasis plus genetic co-expression with defined readout, single lab\",\n      \"pmids\": [\"12554670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Biochemical characterization of archaeal Cdc6 (SsoCdc6-1 from Sulfolobus solfataricus): binds ssDNA and dsDNA, has weak ATPase activity, undergoes autophosphorylation; Walker A mutant (K59A) abolishes ATPase and autophosphorylation; SsoCdc6-1 strongly inhibits ATPase and DNA helicase activity of S. solfataricus MCM—the first in vitro evidence of functional Cdc6-MCM interaction.\",\n      \"method\": \"Recombinant protein expression, EMSA, ATPase assay, autophosphorylation assay, Walker A mutagenesis, MCM helicase inhibition assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple in vitro biochemical assays with mutagenesis and reconstituted protein-protein functional interaction, single study\",\n      \"pmids\": [\"12966100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Mitotic CDK Clb2/Cdc28 binds tightly to an N-terminal domain (NTD) of Cdc6 only when the NTD is phosphorylated on CDK consensus sites; Cdc6 in this complex cannot assemble pre-RCs; human CDKs with cyclins A, B, and E also bind phospho-NTD peptides; this Clb2-dependent mechanism contributes to preventing re-replication in vivo.\",\n      \"method\": \"In vitro binding assays with recombinant Clb2 and synthetic phospho-NTD peptides, co-IP, pre-RC assembly assay, in vivo re-replication assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted peptide binding with phosphorylation dependence, in vivo pre-RC assembly assay, and cross-species validation with human CDKs\",\n      \"pmids\": [\"15496876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CDC6 is required for meiotic spindle formation in mouse oocytes; RNAi-mediated knockdown of CDC6 prevents meiotic spindle assembly without affecting resumption of meiosis, revealing a role for CDC6 in spindle organization beyond its established function in DNA replication.\",\n      \"method\": \"RNA interference knockdown of CDC6 in mouse oocytes, meiotic spindle immunofluorescence\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi with specific morphological phenotype, single lab\",\n      \"pmids\": [\"15385409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"S. cerevisiae Cdc6 binds cooperatively with ORC on origin DNA in an ATP-dependent manner, inducing a change in origin binding pattern that requires the Orc1 ATPase; single-particle EM reconstruction shows ORC-Cdc6 forms a ring-shaped complex with dimensions similar to the MCM helicase, predicted to contain six AAA+ subunits.\",\n      \"method\": \"ATP-dependent DNA binding assays, origin mutation analysis, single-particle electron microscopy reconstruction\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical reconstitution plus EM structural analysis, multiple origin mutations tested\",\n      \"pmids\": [\"16228006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Recruitment of CDC6 (as a GAL4-DBD fusion) to a defined DNA array is sufficient to create a functional artificial origin of replication in mammalian cells; the ATPase domain of human Cdc6 is functionally important; N-terminal segments of ORC1/ORC2 are dispensable in this assay.\",\n      \"method\": \"GAL4-DBD fusion tethering assay, replication assay with geminin inhibition, ATPase domain mutant analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional origin assay with domain mutants, single lab\",\n      \"pmids\": [\"16322558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"p53 activation by DNA damage enhances Cdc6 destruction via the APC; this destruction is triggered by inhibition of CDK2-mediated phosphorylation of CDC6 at serine 54; suppression of p53 stabilizes Cdc6, leading to more replicating cells—an effect reversed by reducing Cdc6 levels.\",\n      \"method\": \"DNA damage treatment, CDK2 inhibition, p53 knockdown, Cdc6 stability assays, phosphorylation-site mutagenesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation-site mutagenesis with p53-pathway epistasis, single lab\",\n      \"pmids\": [\"16055707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"High levels of Cdc6 repress transcription of the INK4/ARF locus (p15INK4b, ARF, p16INK4a) through a replication origin (RD-INK4/ARF) that assembles Cdc6/Orc2/MCM complexes; Cdc6 overexpression recruits histone deacetylases and induces heterochromatinization of this locus; Cdc6 has cellular immortalization and transformation activities in cooperation with Ras.\",\n      \"method\": \"ChIP for Cdc6/Orc2/MCM at INK4/ARF locus, RNAi-induced heterochromatinization, HDAC recruitment assay, transformation assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP, HDAC recruitment, and functional transformation assays with multiple orthogonal methods in single study\",\n      \"pmids\": [\"16572177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cdc6 depletion during S phase (not G1) slows DNA replication and leads to mitotic lethality; Cdc6-depleted S-phase cells show fewer newly fired origins but active established replication forks; loss of Cdc6 in S phase fails to activate Chk1 kinase.\",\n      \"method\": \"RNAi-mediated depletion in synchronous G1 vs S-phase cells, origin firing analysis, Chk1 activation assay, mitotic outcome analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi in synchronized cell populations with multiple functional readouts, single lab\",\n      \"pmids\": [\"16439999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Caspase-3 cleaves Cdc6 at D290/S and D442/G sites during apoptosis; resulting truncated fragments (p32-tCdc6 and p49-tCdc6) promote apoptosis, perturb MCM2 (but not Orc2) chromatin loading, and activate ATM and ATR kinases with kinetics consistent with Chk1/2 phosphorylation.\",\n      \"method\": \"In vitro caspase-3 cleavage mapping, ectopic expression of tCdc6 fragments, chromatin loading assay for MCM2/Orc2, ATM/ATR kinase activation assay, siRNA for ATM/ATR\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical cleavage site mapping with functional ATM/ATR activation assay and MCM loading, single lab\",\n      \"pmids\": [\"16801388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Cdc6 ATPase activity is activated by ORC, regulates ORC-Cdc6 complex stability, and is suppressed by origin DNA; specific origin DNA sequences (particularly the A element) down-regulate Cdc6 ATPase, resulting in stable ORC-Cdc6-DNA complex formation competent for MCM loading; on non-origin DNA, Cdc6 ATPase promotes Cdc6 dissociation.\",\n      \"method\": \"In vitro ATPase assay with ORC and various DNA sequences, ATPase mutants, ORC-Cdc6 complex stability assays on different DNAs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro ATPase and complex stability assays with multiple DNA substrates and mutants, mechanistic model\",\n      \"pmids\": [\"17314092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Cdc6 stability after UV irradiation or MMS-induced DNA damage is regulated by the HECT-family ubiquitin E3 ligase Huwe1 (Mule/ARF-BP1); Cdc6 directly binds Huwe1; Huwe1 polyubiquitinates Cdc6 in vitro; this pathway is independent of p53, Cdc6 CDK-phosphorylation sites, and APC-Cdh1; it is conserved in yeast (Tom1 ortholog) and is associated with Cdc6 release from chromatin.\",\n      \"method\": \"Co-IP of Cdc6 with Huwe1, in vitro ubiquitination assay, Huwe1 knockdown, Tom1 deletion in yeast, UV/MMS treatment\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitination, co-IP, knockdown in human cells plus yeast ortholog validation; multiple orthogonal methods\",\n      \"pmids\": [\"17567951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Cdc6 represses CDH1 (E-cadherin) transcription by binding to E-boxes in the CDH1 promoter, causing dissociation of the insulator CTCF, displacement of histone variant H2A.Z, and promoter heterochromatinization; mutational analysis identifies the Walker B motif and C-terminal region of Cdc6 as essential for this transcriptional suppression; CTCF displacement also activates adjacent replication origins.\",\n      \"method\": \"ChIP for Cdc6, CTCF, H2A.Z at CDH1 promoter, overexpression of Cdc6 in epithelial cells, mutational analysis of Walker B and C-terminal domain, replication origin activation assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP with mutagenesis and functional readouts (transcription and origin activation), multiple orthogonal methods\",\n      \"pmids\": [\"22201124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cryo-EM structure of S. cerevisiae ORC-Cdc6 on ARS1 origin DNA shows Cdc6 binding changes ORC conformation, particularly reorienting the Orc1 N-terminal BAH domain; a single Cdc6 extends the ORC footprint on origin DNA from both ends; the crescent-like ORC bends and wraps DNA.\",\n      \"method\": \"Single-particle cryo-EM, docking of archaeal Orc1/Cdc6 crystal structure, DNase I footprinting\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structural analysis with footprinting validation, single lab\",\n      \"pmids\": [\"22405012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cdc6 obstructs apoptosome assembly by forming stable complexes with cytochrome c-activated Apaf-1 monomers; this function depends on Cdc6's ATPase domain but not its cyclin-binding motif; in proliferating cells, Cdc6 suppresses seemingly unintended apoptosis while promoting cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation of Cdc6 with Apaf-1, apoptosome assembly assay, ATPase domain mutant and cyclin-binding motif mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with domain mutant analysis and functional apoptosome assay, single lab\",\n      \"pmids\": [\"22493447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"A yeast GSK-3 kinase homolog Mck1 promotes Cdc6 degradation by phosphorylating Cdc6 at Threonine-368 (a GSK-3 consensus site), leading to SCF(CDC4)-dependent proteolysis; mck1 deletion stabilizes Cdc6 in late S phase and mitosis; Mck1-dependent Cdc6 degradation is required to prevent DNA re-replication.\",\n      \"method\": \"Deletion analysis of MCK1, protein stability assays, phosphorylation-site mutagenesis (T368A), SCF genetic epistasis, DNA content analysis for re-replication\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation-site mutagenesis with genetic epistasis and re-replication readout, single lab\",\n      \"pmids\": [\"23236290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"During pre-RC assembly, ORC-Cdc6 forms an intermediate ORC-Cdc6-MCM2-7 (OCM) complex that is competent for MCM2-7 dimerization; the initial ORC-Cdc6-Cdt1-MCM2-7 (OCCM) complex is not competent for dimerization; MCM2-7 dimerization is a limiting, Cdc6-dependent step in pre-RC formation.\",\n      \"method\": \"Biochemical reconstitution of pre-RC assembly, MCM2-7 hexamer-interface mutants, complex analysis by EM, salt-sensitivity assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with defined mutants and EM validation, identification of a specific assembly intermediate, single lab\",\n      \"pmids\": [\"24234446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A PIP-box in the N-terminus of Cdc6 mediates APC/C-CDH1-independent degradation of nuclear Cdc6 at the G1-S transition and during S phase via the CRL4-Cdt2 complex, preventing nuclear Cdc6 re-accumulation; Cdk1 contributes to nuclear export of Cdc6 at the S-to-G2 transition.\",\n      \"method\": \"PIP-box mutagenesis, Cdt2 knockdown, cell cycle synchronization, nuclear/cytoplasmic fractionation, Cdc6 stability assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PIP-box mutagenesis with Cdt2 depletion and fractionation, single lab\",\n      \"pmids\": [\"24434580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cdc6 ATPase activity is required for Cdc6 disengagement from the pre-RC after MCM helicase loading, not for MCM loading per se; an ATPase-defective Cdc6-E224Q mutant supports MCM loading but cells remain blocked in G1; degradation of Cdc6-E224Q after MCM loading restores apparently normal S phase, demonstrating that Cdc6 must disengage post-loading to allow helicase activation.\",\n      \"method\": \"Purified protein reconstitution of MCM loading with Cdc6 ATPase mutants, in vivo MCM chromatin association, conditional Cdc6 degradation experiments, S phase progression assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro loading with ATPase mutant plus in vivo conditional degradation rescue, mechanistically conclusive\",\n      \"pmids\": [\"26305410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SCF(Cyclin F) ubiquitin ligase complex targets CDC6 for proteasomal degradation late in the cell cycle through defined sequence motifs; absence of Cyclin F or expression of a stable CDC6 mutant promotes DNA re-replication and genome instability in cells lacking Geminin.\",\n      \"method\": \"Co-IP of CDC6 with Cyclin F, ubiquitination assay, stable CDC6 mutant expression, re-replication assay by flow cytometry, Geminin depletion\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, ubiquitination assay, and functional re-replication assay with stable mutant, multiple orthogonal methods\",\n      \"pmids\": [\"26818844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ORC1 represses Cyclin E gene (CCNE1) transcription by binding RB, the histone methyltransferase SUV39H1, and its repressive H3K9me3 mark; in contrast, CDC6 binds Cyclin E-CDK2 and removes RB from ORC1 in a feedback loop, thereby hyper-activating CCNE1 transcription; ORC1 and CDC6 thus have opposing effects on cell cycle commitment.\",\n      \"method\": \"Co-IP of ORC1 with RB and SUV39H1, ChIP for H3K9me3, promoter reporter assay, CDC6 overexpression removing RB from ORC1\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and ChIP with functional reporter assay, single lab\",\n      \"pmids\": [\"27458800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) at 3.9 Å shows Cdc6 winged-helix domain and positively charged loops contact origin DNA; flexible Mcm2-7 winged-helix domains engage ORC-Cdc6; DNA passes through both the ORC-Cdc6 and Mcm2-7 rings; Cdt1 embraces Mcm2, Mcm4, and Mcm6 comprising nearly half the hexamer.\",\n      \"method\": \"Cryo-EM structure determination at 3.9 Å resolution, crosslinking mass spectrometry\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic resolution cryo-EM structure of the complete loading intermediate with crosslinking MS validation\",\n      \"pmids\": [\"28191893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cdc6 is recruited to centrioles via Cyclin A and negatively regulates centrosome duplication by binding Sas-6, inhibiting stable Sas-6/STIL complex formation; Plk4 phosphorylates Cdc6 to disrupt Cdc6-Sas-6 interaction, counteracting Cdc6's inhibitory role on centrosome duplication; Cdc6 and Plk4 thus antagonistically control centrosome number.\",\n      \"method\": \"Co-IP of Cdc6 with Sas-6 and STIL, immunofluorescence for Cdc6 at centrosomes, Plk4 phosphorylation of Cdc6, centrosome duplication assay with Cdc6 mutants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with kinase substrate identification and functional centrosome assay, single lab\",\n      \"pmids\": [\"28447620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM structures of Drosophila ORC with and without Cdc6 reveal that Orc1 and Orc4 constitute the primary DNA binding site; a Walker B loop of Orc1 contacts DNA, allosterically coupling DNA binding to the ATPase site; Cdc6 binding promotes DNA bending which facilitates MCM2-7 loading in vitro.\",\n      \"method\": \"Cryo-EM structure of Drosophila ORC ± Cdc6 on DNA, biochemical DNA-binding and MCM loading assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structural determination combined with functional biochemical assays, single study\",\n      \"pmids\": [\"32848132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Two ORC-Cdc6-Cdt1-Mcm2-7 loading intermediates prior to DNA insertion were captured by cryo-EM: 'semi-attached OCCM' where Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 without the main body docking, and 'pre-insertion OCCM' where Mcm2-7 docks and origin DNA is bent adjacent to the Mcm2-Mcm5 open gate; molecular simulations show dynamic transition to the fully loaded state.\",\n      \"method\": \"Cryo-EM of loading intermediates (Mcm6-WHD truncation to slow reaction), molecular dynamics simulations\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structural intermediates with computational mechanistic analysis, multi-lab collaboration\",\n      \"pmids\": [\"32669428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of S. cerevisiae ORC-Cdc6 on ARS1 origin DNA at 3.3 Å reveals that Cdc6 contributes to origin DNA recognition via its winged-helix domain and initiator-specific motif; Cdc6 binding rearranges an α-helix in the Orc1 AAA+ domain and the Orc2 WHD, activating the Cdc6 ATPase and forming three Mcm2-7 recruitment sites absent in ORC alone.\",\n      \"method\": \"Cryo-EM at 3.3 Å resolution\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM revealing atomic-level mechanism of Cdc6-induced ORC activation\",\n      \"pmids\": [\"34162887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Multiple short linear motifs (SLiMs) in the intrinsically disordered region (IDR) of CDC6 mediate cyclin-CDK-dependent and -independent interactions; the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interaction in G1; the CDC6 IDR regulates ORC1 self-interaction and controls ORC1 protein levels; Protein Phosphatase 1 binds ORC1 IDR causing de-phosphorylation at mitotic exit.\",\n      \"method\": \"Co-IP of CDC6 SLiM mutants with cyclins and ORC1, CDK2 kinase assays, cell cycle synchronization with protein level measurements\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SLiM mutagenesis with co-IP and kinase assays, single lab\",\n      \"pmids\": [\"33761311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The deubiquitinase OTUD6A interacts with, deubiquitinates (removing K6-, K33-, and K48-linked polyubiquitin chains), and stabilizes CDC6 protein; OTUD6A promotes tumor cell proliferation and chemoresistance via CDC6 upregulation; OTUD6A-CDC6 axis is conserved in an in vivo bladder cancer model.\",\n      \"method\": \"Proteome-wide DUB screening, co-IP of OTUD6A with CDC6, in vitro deubiquitination assay, protein half-life assay, conditional Otud6a KO mouse model, xenograft model\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro deubiquitination reconstitution, co-IP, plus in vivo mouse KO and xenograft models; multiple orthogonal methods\",\n      \"pmids\": [\"38685067\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CDC6 is an AAA+ ATPase that, upon binding to ORC on replication origins in G1, forms a ring-shaped ORC-Cdc6 complex that recruits Cdt1-bound Mcm2-7 hexamers (OCCM intermediate), loads the MCM2-7 double hexamer, and then disengages via its own ATPase activity to permit helicase activation; its abundance and localization are tightly controlled through cell cycle-regulated E2F-dependent transcription, Cyclin A/CDK2-mediated phosphorylation-driven nuclear export, and sequential ubiquitin-mediated proteolysis by APC/C-CDH1 (G1/quiescence), SCF-Cdc4 (G1/S), SCF-Cyclin F (late cell cycle), and Huwe1 (DNA damage), while its stabilization by the deubiquitinase OTUD6A can promote oncogenesis; beyond DNA replication licensing, CDC6 inhibits mitotic CDKs to couple S phase completion to mitotic entry, represses tumor suppressor loci (INK4/ARF, CDH1) through chromatin remodeling, inhibits apoptosome assembly via Apaf-1 binding, and is cleaved by caspase-3 during apoptosis to generate pro-apoptotic nuclear fragments.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CDC6 is an essential AAA+ ATPase that licenses eukaryotic DNA replication origins by cooperating with ORC to load the MCM2-7 replicative helicase, and it has been conserved from archaea and yeast to humans as a master regulator coupling origin firing to cell cycle progression [#0, #4, #31]. Cdc6 binds ORC cooperatively on origin DNA in an ATP-dependent manner to form a ring-shaped, six-AAA+-subunit ORC-Cdc6 complex that bends origin DNA, rearranges ORC (including the Orc1 AAA+ domain and BAH/winged-helix elements), and creates the MCM2-7 recruitment sites absent from ORC alone [#31, #40, #50, #52]. Cdc6's own winged-helix and initiator-specific motifs contact origin DNA within the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) loading intermediate, through which DNA is threaded; the reaction proceeds via defined semi-attached and pre-insertion intermediates and an ORC-Cdc6-MCM (OCM) state competent for MCM2-7 dimerization [#43, #48, #51]. Cdc6 ATPase activity, activated by ORC and suppressed by origin DNA, is required not for loading itself but for Cdc6 disengagement from the pre-RC after helicase loading, a step essential for subsequent helicase activation [#37, #45]. Cdc6 abundance, localization, and activity are tightly cell-cycle-controlled: human CDC6 transcription is driven by E2F factors and CDC6 cooperates with cyclin E to drive S-phase entry [#7, #9, #23], while Cyclin A/CDK2 phosphorylation of N-terminal CDK sites drives nuclear-to-cytoplasmic relocalization and destruction of non-chromatin-bound Cdc6 to prevent re-replication [#12, #16]. Cdc6 is degraded by sequential ubiquitin pathways—APC/C-CDH1 in G1 and quiescence, SCF-Cdc4, CRL4-Cdt2, SCF-Cyclin F late in the cycle, and the HECT ligase Huwe1 after DNA damage—and CDK/Clb binding to phosphorylated Cdc6 also inhibits pre-RC assembly [#15, #29, #38, #44, #46]. Beyond licensing, Cdc6 restrains mitotic entry by binding and inhibiting CDK complexes and acting cooperatively with Sic1 [#2, #21, #27], represses the INK4/ARF and CDH1 loci through origin-coupled chromatin remodeling and HDAC recruitment with transforming activity [#34, #39], and obstructs apoptosome assembly by sequestering Apaf-1, while caspase-3 cleavage of Cdc6 during apoptosis generates nuclear pro-apoptotic, replication-inhibitory fragments [#41, #25, #26]. The deubiquitinase OTUD6A stabilizes CDC6 to promote tumor proliferation and chemoresistance [#54].\",\n  \"teleology\": [\n    {\n      \"year\": 1989,\n      \"claim\": \"Established CDC6 as an essential, conserved nucleotide-binding protein required for S-phase entry, defining the gene's core role before any mechanism was known.\",\n      \"evidence\": \"Complementation cloning, gene disruption, and sequencing in S. cerevisiae\",\n      \"pmids\": [\"2656692\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical activity demonstrated\", \"Molecular role at origins unknown\"]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Showed CDC6 expression is cell-cycle periodic, peaking at G1/S, framing it as a transcriptionally gated licensing factor.\",\n      \"evidence\": \"Synchronized yeast cultures, Northern blotting, promoter analysis\",\n      \"pmids\": [\"2246267\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factors driving periodicity not yet defined\", \"Protein-level regulation unaddressed\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Demonstrated that Cdc6 is required for replication initiation and that its absence uncouples replication from mitosis, revealing a checkpoint-like coupling function.\",\n      \"evidence\": \"Yeast genetics, synchronization, FISH, transcription factor mutants\",\n      \"pmids\": [\"7641697\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of mitotic coupling unresolved\", \"Direct origin role not yet shown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Identified Cdc6 as forming origin pre-replicative complexes distinct from ORC and as a binding partner/inhibitor of B-type cyclin/Cdc28 kinase, linking origin licensing to CDK control.\",\n      \"evidence\": \"Genomic footprinting at yeast origins; co-IP, pulldown, in vitro kinase assays, deletion mutants\",\n      \"pmids\": [\"8978693\", \"8930895\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of CDK inhibition not structurally defined\", \"How Cdc6 nucleates MCM loading unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Extended the model to humans: E2F-driven transcription, ORC1 and cyclin-CDK association, NLS-independent nuclear localization, and antibody-blocked replication established CDC6 as a conserved human licensing factor.\",\n      \"evidence\": \"Promoter-reporter, in vivo footprinting, antibody microinjection, fractionation, co-IP, NLS mutagenesis in human cells\",\n      \"pmids\": [\"9520412\", \"9566895\", \"9774682\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymatic requirement not yet tested in human protein\", \"Mechanism of S-phase nuclear elimination unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined Cdc6 ATPase activity as essential for replication and showed Cyclin A/CDK2 phosphorylation controls its localization, establishing both the catalytic requirement and the re-replication safeguard.\",\n      \"evidence\": \"Recombinant ATP binding/hydrolysis with Walker A/B mutants and microinjection; in vitro kinase, domain mapping, fractionation, immunofluorescence\",\n      \"pmids\": [\"10436018\", \"9889196\", \"9857179\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Step in licensing requiring ATPase not yet pinpointed\", \"How phosphorylation triggers export mechanistically unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Resolved that ubiquitin-mediated proteolysis (APC/C-CDH1 in G1/quiescence; SCF-Cdc4 in yeast) and Cyclin A-CDK2 phosphorylation selectively destroy non-chromatin-bound Cdc6, preventing re-licensing.\",\n      \"evidence\": \"In vitro APC/CDH1 ubiquitination, depletion, D-box/KEN-box mutagenesis; chromatin fractionation with recombinant Cyclin A-CDK2; yeast cdc4 epistasis\",\n      \"pmids\": [\"10995389\", \"10806104\", \"10085159\", \"9442103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination between distinct degradation pathways unresolved\", \"Chromatin-bound vs soluble pool discrimination mechanism incomplete\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Crystallography of an archaeal Cdc6 ortholog revealed the AAA+ fold with a DNA-binding winged-helix domain, providing the structural framework for origin recognition and showing both domains are functionally required.\",\n      \"evidence\": \"X-ray crystallography (2.0 A), WH and ATPase mutagenesis with S. pombe functional assays\",\n      \"pmids\": [\"11030343\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Eukaryotic complex architecture not yet visualized\", \"How WH domain engages origin DNA in ORC context unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed Cdc6 directly inactivates mitotic CDKs (with Sic1) and recruits Cyclin E-CDK2 to chromatin via RXL motifs, formalizing dual roles in licensing and mitotic restraint.\",\n      \"evidence\": \"Genetic CDK-domain deletion and epistasis, co-IP, in vitro CDK assays; Xenopus extract chromatin recruitment with RXL/MRAIL mutagenesis\",\n      \"pmids\": [\"11460169\", \"11257126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of Cdc6 vs Sic1 to mitotic exit unclear\", \"How chromatin-localized CDK2 acts on licensing not fully defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstrated Cdc6 alone suffices to load MCM and license origins in quiescent cells in an ATPase-dependent, cyclin E-CDK2-synergistic manner, and is required to re-establish replication competence in meiotic oocytes.\",\n      \"evidence\": \"Adenoviral wild-type/mutant Cdc6 with MCM chromatin assays; Xenopus oocyte Cdc6 protein injection with synthesis inhibition\",\n      \"pmids\": [\"11805305\", \"12384699\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of MCM loading not defined\", \"Mechanism of CDK isoform specificity unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Provided first reconstituted Cdc6-MCM functional interaction (archaeal) and showed human Cdc6 overexpression in G2 blocks mitosis via a Chk1-dependent checkpoint, reinforcing replication-mitosis coupling.\",\n      \"evidence\": \"Archaeal recombinant biochemistry (EMSA, ATPase, MCM inhibition, Walker A mutant); G2 overexpression with UCN-01 and Cdc25 epistasis\",\n      \"pmids\": [\"12966100\", \"12554670\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of G2 checkpoint role unclear\", \"Direct human Cdc6-MCM contacts not yet structurally defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Visualized the ring-shaped ORC-Cdc6 complex and showed Cdc6 tethering creates an artificial mammalian origin, establishing Cdc6 as the architectural trigger for origin assembly.\",\n      \"evidence\": \"ATP-dependent DNA binding, single-particle EM; GAL4-DBD tethering replication assay with ATPase mutants\",\n      \"pmids\": [\"16228006\", \"16322558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic detail of DNA engagement not yet resolved\", \"MCM recruitment geometry unknown at this resolution\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed a non-licensing oncogenic function: high Cdc6 represses the INK4/ARF tumor suppressor locus via origin-coupled HDAC recruitment and heterochromatinization, with Ras-cooperative transforming activity.\",\n      \"evidence\": \"ChIP for Cdc6/Orc2/MCM at INK4/ARF, HDAC recruitment, RNAi heterochromatinization, transformation assays\",\n      \"pmids\": [\"16572177\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality across other loci not yet established\", \"Link between origin function and repression mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the regulatory logic of Cdc6 ATPase—activated by ORC, suppressed by origin DNA—to stabilize productive ORC-Cdc6-DNA complexes at origins while dissociating Cdc6 from non-origin DNA.\",\n      \"evidence\": \"In vitro ATPase and complex-stability assays with multiple DNA substrates and mutants; co-IP, in vitro ubiquitination and Huwe1 knockdown for damage-induced turnover\",\n      \"pmids\": [\"17314092\", \"17567951\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coupling of ATPase state to MCM loading step not yet resolved\", \"Damage-induced chromatin release mechanism partly defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed Cdc6 represses CDH1 (E-cadherin) by E-box binding, CTCF dissociation, and H2A.Z displacement, extending its chromatin-repressive and origin-activating activities to an EMT-relevant locus.\",\n      \"evidence\": \"ChIP for Cdc6/CTCF/H2A.Z, Walker B and C-terminal mutagenesis, origin activation assay\",\n      \"pmids\": [\"22201124\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo tumor relevance not directly tested here\", \"How replication and repression activities are coordinated unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided cryo-EM of ORC-Cdc6 on origin DNA and revealed Cdc6's anti-apoptotic function via Apaf-1 sequestration, broadening its role to cell survival.\",\n      \"evidence\": \"Single-particle cryo-EM with footprinting; co-IP with Apaf-1, apoptosome assembly assay with domain mutants\",\n      \"pmids\": [\"22405012\", \"22493447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Apaf-1 interaction structurally undefined\", \"Physiological threshold of anti-apoptotic activity unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified the ORC-Cdc6-MCM (OCM) intermediate as the Cdc6-dependent, dimerization-competent step distinguishing it from the earlier OCCM, defining the rate-limiting transition in helicase loading.\",\n      \"evidence\": \"Biochemical pre-RC reconstitution, MCM interface mutants, EM, salt-sensitivity assays\",\n      \"pmids\": [\"24234446\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational basis of dimerization competence not yet atomic\", \"Timing relative to Cdt1 release unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated definitively that Cdc6 ATPase is required for post-loading disengagement of Cdc6, not MCM loading itself—resolving a long-standing question about the catalytic step.\",\n      \"evidence\": \"Reconstituted loading with Cdc6-E224Q ATPase mutant plus in vivo conditional degradation rescue\",\n      \"pmids\": [\"26305410\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How disengagement enables helicase activation mechanistically unclear\", \"Structural state of disengaged complex undefined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Added SCF-Cyclin F as a late-cycle CDC6 degradation pathway guarding against re-replication, and showed ORC1/CDC6 form an opposing transcriptional circuit at CCNE1, integrating licensing with cell-cycle commitment.\",\n      \"evidence\": \"Co-IP, ubiquitination, re-replication flow cytometry with stable mutant; ORC1-RB-SUV39H1 co-IP/ChIP with CDC6 RB-displacement assay\",\n      \"pmids\": [\"26818844\", \"27458800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Redundancy among degradation pathways not quantified\", \"Direct demonstration of feedback circuit in normal proliferation incomplete\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Near-atomic cryo-EM of the OCCM and discovery of Cdc6 at centrioles revealed both the structural basis of origin licensing and a moonlighting role limiting centrosome duplication.\",\n      \"evidence\": \"Cryo-EM of OCCM at 3.9 A with crosslinking MS; co-IP with Sas-6/STIL, centrosome assays, Plk4 phosphorylation\",\n      \"pmids\": [\"28191893\", \"28447620\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DNA insertion step not captured\", \"Physiological extent of centrosome regulation unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Captured loading intermediates and DNA bending mechanics by cryo-EM and MD, showing how Cdc6 binding promotes DNA bending and stepwise MCM2-7 engagement toward the loaded state.\",\n      \"evidence\": \"Cryo-EM of Drosophila ORC +/- Cdc6 and yeast semi-attached/pre-insertion OCCM with molecular dynamics\",\n      \"pmids\": [\"32848132\", \"32669428\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full DNA insertion and gate closure dynamics still inferred\", \"How ATPase activity drives these transitions not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved at 3.3 A how Cdc6 contributes to origin recognition and allosterically activates its own ATPase by rearranging ORC, and defined CDC6 disordered-region SLiMs governing cyclin-CDK and ORC1 interactions.\",\n      \"evidence\": \"Cryo-EM at 3.3 A; SLiM mutagenesis with co-IP and CDK2 assays\",\n      \"pmids\": [\"34162887\", \"33761311\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of ATPase activation during loading still modeled, not observed\", \"Functional hierarchy of individual SLiMs incomplete\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified OTUD6A as a deubiquitinase that stabilizes CDC6 to drive tumor proliferation and chemoresistance, providing a disease-relevant post-translational control point.\",\n      \"evidence\": \"DUB screen, co-IP, in vitro deubiquitination, half-life assays, conditional KO mouse, xenograft bladder cancer model\",\n      \"pmids\": [\"38685067\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether OTUD6A acts on chromatin-bound vs soluble CDC6 unknown\", \"Generality across cancer types not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Cdc6 ATPase-driven disengagement is mechanically coupled to helicase activation, and how its licensing function is integrated with its chromatin-repressive, anti-apoptotic, and centrosomal roles in a single cell, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the disengaged post-loading state\", \"Mechanism connecting origin function to transcriptional repression unclear\", \"How moonlighting functions are temporally partitioned from licensing unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [3, 10, 23, 37, 45]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [3, 10, 37]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [18, 28, 48, 52]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 21, 29, 41]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [34, 39, 47]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 12, 16, 26]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [5, 14, 34]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12, 16]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [49]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [4, 11, 23, 43, 45, 48]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 21, 27, 12]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [34, 39]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [25, 26, 41]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [15, 38, 46, 54]}\n    ],\n    \"complexes\": [\"ORC-Cdc6\", \"ORC-Cdc6-Cdt1-Mcm2-7 (OCCM)\", \"ORC-Cdc6-Mcm2-7 (OCM)\", \"pre-replicative complex\"],\n    \"partners\": [\"ORC1\", \"MCM2-7\", \"CDT1\", \"CCNA2\", \"CCNE1\", \"HUWE1\", \"OTUD6A\", \"APAF1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}