{"gene":"CDT1","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1994,"finding":"CDT1 (cdt1) was identified as an essential target gene of the fission yeast Cdc10/Sct1 transcription factor; cells carrying a null allele of cdt1 are defective in DNA replication but still initiate mitotic events, establishing cdt1 as essential for the S phase–mitosis dependency relationship.","method":"Immunoprecipitation-PCR to isolate Cdc10-bound genomic sequences; null allele analysis showing DNA replication defect without mitotic block","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null allele with defined replication phenotype, replicated across subsequent studies in multiple organisms","pmids":["8313888"],"is_preprint":false},{"year":2000,"finding":"Fission yeast Cdt1 is located in the nucleus, cooperates with Cdc18/Cdc6 to load the MCM protein Cdc21 onto chromatin at the end of mitosis, and both factors are required to initiate DNA replication; overexpression of both together drives extensive re-replication.","method":"Re-replication assay in S. pombe; chromatin fractionation; nuclear localization by microscopy; genetic epistasis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (chromatin fractionation, re-replication assay, genetic cooperation), replicated in subsequent studies","pmids":["10766248"],"is_preprint":false},{"year":2000,"finding":"Geminin binds tightly to Cdt1, and excess Cdt1 reverses geminin-mediated inhibition of DNA replication in cell-free Xenopus extracts, establishing that geminin inhibits replication by targeting Cdt1.","method":"Cell-free Xenopus egg extract DNA replication assay; co-immunoprecipitation; rescue by excess Cdt1","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro replication assay with direct rescue experiment; independently replicated by multiple labs","pmids":["11125146"],"is_preprint":false},{"year":2001,"finding":"RLF-B (replication licensing factor B) was purified and shown to correspond to Cdt1; geminin inhibits RLF-B/Cdt1 activity, and immunodepletion of geminin from metaphase Xenopus extracts allows assembly of licensed replication origins.","method":"Protein purification from Xenopus egg extracts; immunodepletion; replication licensing assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical purification of activity, immunodepletion rescue, replicated independently","pmids":["11175741"],"is_preprint":false},{"year":2001,"finding":"Human Cdt1 (hCdt1) is a nuclear protein present only in G1; after S-phase onset its levels decrease via proteasomal proteolysis (not transcriptional changes), and hCdt1 can associate with geminin.","method":"Cell cycle synchronization; immunoblotting; proteasome inhibitor stabilization; co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple methods (synchronization, proteasome inhibition, co-IP); replicated extensively","pmids":["11555648"],"is_preprint":false},{"year":2002,"finding":"Budding yeast Cdt1 (Tah11/Sid2) is required for pre-RC assembly; depletion blocks Mcm2 chromatin loading and replication initiation but not elongation; Cdt1 accumulates in the nucleus during G1 and is excluded later by CDKs, and its nuclear accumulation is interdependent with Mcm2-7.","method":"Conditional depletion; chromatin fractionation; co-immunoprecipitation; cell cycle analysis","journal":"Nature cell biology / Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — independent papers (PMID 11836525 and 11967159) with depletion, chromatin fractionation, co-IP","pmids":["11836525","11967159"],"is_preprint":false},{"year":2002,"finding":"Mouse Cdt1 binds geminin through its central region (aa 177–380) and MCM6 through its C-terminal region (aa 407–477); Cdt1 also has intrinsic sequence-, strand-, and conformation-independent DNA binding activity that overlaps the geminin binding domain and is inhibited by geminin.","method":"Yeast two-hybrid; in vitro binding assays with purified proteins; domain deletion mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — purified proteins, domain mapping, in vitro binding; single lab but multiple orthogonal methods","pmids":["12192004"],"is_preprint":false},{"year":2002,"finding":"Human Cdt1 is essential for DNA replication in intact human cells; it functions during the pre-RC formation window and is required for loading MCM proteins onto chromatin.","method":"Antibody microinjection into human cells; MCM chromatin loading assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct loss-of-function by antibody microinjection with defined MCM loading readout; replicated by subsequent studies","pmids":["11896191"],"is_preprint":false},{"year":2003,"finding":"The SCF(Skp2) complex ubiquitinates Cdt1 in a phosphorylation-dependent manner; Skp2 specifically interacts with Cdt1, and downregulation of Skp2 or disruption of the Cdt1–Skp2 interaction stabilizes and accumulates Cdt1.","method":"Co-immunoprecipitation; in vitro and in vivo ubiquitination assay; siRNA knockdown; dominant-negative disruption","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination reconstitution, in vivo co-IP, KD phenotype; replicated by multiple labs","pmids":["12840033"],"is_preprint":false},{"year":2003,"finding":"Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain; geminin inhibits Cdt1 by blocking its binding to both Mcm2 and Cdc6; the Cdt1–Mcm2 association is regulated by cell growth and induced by Cdc6 expression.","method":"Co-immunoprecipitation; ectopic expression in quiescent cells; domain mapping","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP, ectopic expression, single lab but multiple approaches","pmids":["14672932"],"is_preprint":false},{"year":2003,"finding":"CDT1 is rapidly degraded after UV or gamma-irradiation via the CUL4-ROC1 E3 ubiquitin ligase and the COP9 signalosome; loss of either complex completely suppresses CDT1 proteolysis; CDT1 is specifically polyubiquitinated by CUL4 complexes.","method":"siRNA knockdown of CUL4, ROC1, CSN components; in vivo ubiquitination assay; irradiation-induced degradation assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockdown with defined proteolysis phenotype, in vivo ubiquitination, replicated by multiple labs","pmids":["14578910"],"is_preprint":false},{"year":2004,"finding":"Crystal structure of the mouse geminin–Cdt1 complex reveals that a coiled-coil dimer of geminin interacts with both N-terminal and C-terminal portions of Cdt1; the primary interface involves steric complementarity with Pro181, Ala182, Tyr183, Phe186, and Leu189 of Cdt1, and the C-terminal region of geminin sterically blocks MCM access to Cdt1.","method":"X-ray crystallography; mutagenesis; biochemical binding assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis validation","pmids":["15286659"],"is_preprint":false},{"year":2004,"finding":"DDB1 associates stoichiometrically with CUL4A in vivo; recombinant DDB1 and CDT1 bind directly in vitro; DDB1 bridges CDT1 to CUL4A in vivo; silencing DDB1 prevents UV-induced CDT1 degradation and CUL4A-mediated CDT1 ubiquitination in vitro, establishing DDB1 as the substrate adaptor for CUL4A-mediated CDT1 ubiquitination.","method":"Co-immunoprecipitation; in vitro direct binding with purified recombinant proteins; in vitro ubiquitination assay; siRNA knockdown","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins plus in vivo co-IP and knockdown; replicated by multiple labs","pmids":["15448697"],"is_preprint":false},{"year":2004,"finding":"Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the first round of DNA replication in Xenopus egg extracts; degradation requires Cdk2/Cyclin E, Cdc45, RPA, and polymerase alpha (i.e., replication initiation); Cdt1 is ubiquitinated on chromatin.","method":"Xenopus egg extract replication assay; immunodepletion of replication factors; ubiquitination assay on chromatin","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — cell-free reconstitution with defined factor depletions, chromatin ubiquitination assay","pmids":["15598982"],"is_preprint":false},{"year":2004,"finding":"Human Cdt1 is phosphorylated by Cdk2 and Cdk4; phosphorylation at a cyclin-binding motif promotes Skp2 binding and subsequent SCF(Skp2)-mediated degradation; CDK inhibitors (p21, p27) suppress Cdt1 phosphorylation and stabilize it; a quadruple Cdk-binding site mutant fails to bind Skp2 and is more stable.","method":"In vitro kinase assay; co-immunoprecipitation; site-directed mutagenesis; overexpression of CDK inhibitors","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay, mutagenesis, in vivo co-IP; replicated by parallel study PMID 14993212","pmids":["15004027","14993212"],"is_preprint":false},{"year":2004,"finding":"Cyclin A-dependent CDK phosphorylation of Cdt1 (dependent on its cyclin-binding motif) promotes Skp2 binding and degradation, and also inhibits Cdt1 DNA-binding activity in vitro; however, geminin binding to Cdt1 is not affected by phosphorylation; inactivation of Cdk1 in G2/M leads to Cdt1 dephosphorylation and re-binding to chromatin.","method":"In vitro kinase assay; co-immunoprecipitation; in vitro DNA binding assay; synchronization of FT210 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro assays with mutagenesis, in vivo cell cycle validation, single lab","pmids":["14993212"],"is_preprint":false},{"year":2004,"finding":"Geminin forms a parallel coiled-coil homodimer; point mutations disrupting dimerization abolish Cdt1 interaction and replication inhibition; a bipartite interface exists—an array of glutamic acid residues on the coiled-coil surface contacts positive charges in the middle of Cdt1, and an adjoining region independently contacts the N-terminal 100 residues of Cdt1; both interactions are essential for replication inhibition.","method":"X-ray crystallography of geminin; mutagenesis; in vitro replication assay; binding assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure plus mutagenesis plus functional replication assay","pmids":["15260975"],"is_preprint":false},{"year":2004,"finding":"Human Geminin protects CDT1 from proteasome-mediated degradation (by inhibiting its ubiquitination) during S phase and allows CDT1 accumulation during mitosis, establishing a positive role for Geminin in CDT1 stability; CDK1 inhibition in mitosis, not Geminin depletion, is sufficient for premature pre-RC formation in human cells.","method":"siRNA depletion; ubiquitination assay; cell synchronization; immunoblotting","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA with ubiquitination assay, single lab with two orthogonal methods","pmids":["15257290"],"is_preprint":false},{"year":2004,"finding":"Cdt1 proteolysis during S phase in mammalian cells occurs through the N-terminal region (not through geminin binding), is ubiquitin-dependent, and occurs independently of geminin; the N terminus is required for nuclear localization and cyclin A association but is dispensable for geminin association; N-terminus-truncated Cdt1 is stable in S phase.","method":"In vivo ubiquitination assay; siRNA for geminin; temperature-sensitive cell lines; domain truncation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ubiquitination assay, siRNA, domain truncation), replicated","pmids":["15138268"],"is_preprint":false},{"year":2005,"finding":"Replication-dependent proteolysis of Cdt1 in Xenopus egg extracts requires Cdt1 interaction with PCNA through a consensus PIP-box motif; removal of PCNA inhibits Cdt1 destruction; DDB1 (Cul4 E3 component) chromatin loading depends on Cdt1–PCNA binding; mutation of the PIP-box yields stabilized Cdt1 that induces re-replication.","method":"Xenopus egg extract replication assay; PIP-box mutagenesis; PCNA depletion; chromatin fractionation; re-replication assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cell-free reconstitution, mutagenesis, depletion; replicated by multiple labs","pmids":["16362051"],"is_preprint":false},{"year":2005,"finding":"Threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for Skp2 interaction; however, T29 and the Cy motif are not necessary for S-phase proteolysis—a Skp2-independent pathway requiring the first 32 residues of Cdt1 is critical for degradation during S phase and is necessary for normal S-phase progression.","method":"Phosphopeptide mapping; mutagenesis; co-immunoprecipitation; cell cycle analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — phosphopeptide mapping, mutagenesis, domain localization; single lab multiple methods","pmids":["15855168"],"is_preprint":false},{"year":2006,"finding":"Two distinct E3 ubiquitin ligases regulate human Cdt1 proteolysis: DDB1-Cul4 recognizes the first ~10 amino acids (six conserved residues required for PCNA binding); SCF-Skp2 recognizes a Cy-motif region after cyclin E/A-CDK phosphorylation. Both are redundantly required in S-G2; PCNA is essential for Cul4- but not Skp2-directed degradation.","method":"Domain mutagenesis; siRNA co-silencing of Skp2 and Cul4; in vivo ubiquitination assay; PCNA binding assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis, co-silencing, in vivo ubiquitination; replicated by parallel studies","pmids":["16482215"],"is_preprint":false},{"year":2006,"finding":"DCAF2/Cdt2 (a WD40 protein) functions as the substrate receptor of the CUL4-DDB1 E3 ligase to destroy Cdt1 in S phase and after DNA damage in both Xenopus and human cells; depletion of Cdt2 causes rereplication and checkpoint activation; in Xenopus, Cdt2 is recruited to replication forks via Cdt1 and PCNA, where Cdt1 ubiquitylation occurs.","method":"Identification by affinity chromatography/MS; siRNA depletion; cell cycle analysis; Xenopus egg extract assay; chromatin fractionation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS identification, depletion phenotype, Xenopus biochemistry; replicated by multiple labs","pmids":["16949367"],"is_preprint":false},{"year":2006,"finding":"The N-terminal region of Cdt1 contains a PCNA interaction motif required for N-terminal ubiquitination by Cul4/DDB1 after DNA damage and in S phase; the CDK2/Skp2 pathway is not essential for S-phase degradation.","method":"Domain mutagenesis; siRNA knockdown of Cul4, DDB1; in vivo ubiquitination assay; PCNA binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with in vivo ubiquitination, parallel independent confirmation (PMID 16482215, 16362051)","pmids":["16407252"],"is_preprint":false},{"year":2006,"finding":"L2DTL/CDT2 associates with CUL4, DDB1, and PCNA; loss of L2DTL suppresses CDT1 proteolysis after DNA damage; PCNA loss also suppresses CDT1 proteolysis; inactivation of L2DTL dissociates DDB1 from the CUL4 complex in vivo.","method":"Anti-CUL4 affinity chromatography/MS; co-immunoprecipitation; siRNA knockdown; PCNA silencing","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS identification, co-IP, siRNA; replicated by PMID 16949367 and others","pmids":["16861906"],"is_preprint":false},{"year":2006,"finding":"DTL/CDT2 is required for CDT1 downregulation in unperturbed cells and following DNA damage via the CUL4-DDB1 complex; cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels (genetic epistasis).","method":"Zebrafish genetic screen; siRNA in human cells; genetic epistasis (Cdt1 knockdown suppresses Dtl phenotype); co-immunoprecipitation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis plus siRNA plus co-IP across two organisms","pmids":["17085480"],"is_preprint":false},{"year":2007,"finding":"Orc6 directly binds Cdt1 through two regions; ORC lacking Orc6 fails to interact with Cdt1 and cannot load Mcm2-7 onto origin DNA; a fusion protein linking Cdt1 to the Orc6-CTD restores single-round Mcm2-7 loading, showing that dynamic Cdt1–ORC association is required for multiple rounds of loading.","method":"In vitro helicase-loading assay with purified proteins; direct binding assay; Orc6-Cdt1 fusion protein reconstitution","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components, fusion protein rescue, domain mapping","pmids":["18006685"],"is_preprint":false},{"year":2007,"finding":"Cdt1 associates dynamically with chromatin throughout G1 (shown by FRAP); Cdt1 simultaneously binds geminin and chromatin in vivo (contrary to in vitro data) and recruits geminin onto chromatin; chromatin-binding and geminin-binding domains are separable.","method":"Quantitative fluorescence microscopy (FRAP); domain mapping; live-cell imaging in human cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — FRAP with functional domain analysis, single lab but multiple quantitative methods","pmids":["17318181"],"is_preprint":false},{"year":2007,"finding":"APC/C(Cdh1) is a third ubiquitin ligase that controls Cdt1 levels via three destruction boxes in the Cdt1 N-terminus; elimination of these boxes causes strong rereplication and chromosomal damage; Cdt1 also interacts with SNF2H, topoisomerases I and IIα, and GRWD1/WDR28.","method":"Cdt1 affinity chromatography/MS; co-immunoprecipitation; destruction box mutagenesis; rereplication assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with rereplication readout, co-IP, MS; single lab but multiple orthogonal methods","pmids":["18162579"],"is_preprint":false},{"year":2008,"finding":"HBO1 histone acetylase directly interacts with Cdt1; HBO1 associates with replication origins during G1 in a Cdt1-dependent (but Geminin-independent) manner; HBO1 enhances Cdt1-dependent rereplication, establishing HBO1 as a coactivator of the Cdt1 licensing factor at origins.","method":"Co-immunoprecipitation; chromatin immunoprecipitation; rereplication assay; siRNA depletion","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, ChIP, rereplication assay; single lab, multiple methods","pmids":["18832067"],"is_preprint":false},{"year":2008,"finding":"MCM9 forms a stable complex with Cdt1 and is required for Mcm2-7 chromatin loading; MCM9 prevents excess geminin on chromatin during licensing, functioning as an activating linker between Cdt1 and Mcm2-7.","method":"Co-immunoprecipitation; immunodepletion in Xenopus extracts; chromatin fractionation; DNA replication assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, Xenopus depletion/reconstitution, chromatin fractionation; single lab multiple methods","pmids":["18657502"],"is_preprint":false},{"year":2008,"finding":"Cdt1 forms a stable complex with Mcm4/6/7 and Mcm2/3/4/5/6/7 via interaction with Mcm2 and Mcm4/6; Cdt1 stimulates DNA binding and helicase activity of Mcm4/6/7 in vitro; a Cdt1 mutant failing to stimulate helicase activity shows reduced MCM interaction and is deficient in forming the high-MW MCM-Cdt1 assembly.","method":"Glycerol gradient fractionation with purified proteins; in vitro DNA binding and helicase assay; mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins, helicase assay, mutagenesis; single lab","pmids":["18606811"],"is_preprint":false},{"year":2009,"finding":"Cdt1 undergoes acetylation at its N terminus by KAT2B and KAT3B; HDAC11 directly binds and deacetylates Cdt1; acetylation protects Cdt1 from ubiquitylation and proteasomal degradation.","method":"Co-immunoprecipitation; in vitro acetylation assay; HDAC11 binding assay; ubiquitylation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro acetylation reconstitution with single lab, no independent replication identified","pmids":["19276081"],"is_preprint":false},{"year":2009,"finding":"The human Cdt1–Geminin complex can exist as a 'permissive' heterotrimer or an 'inhibitory' heterohexamer; specific Cdt1 residues buried in the heterohexamer are important for licensing; the quaternary-state switch between forms represents a molecular switch between licensing-competent and -defective states.","method":"X-ray crystallography; small-angle X-ray scattering; functional assays in Xenopus and mammalian cells; mutagenesis","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, SAXS, mutagenesis, functional assays in two systems","pmids":["19906994"],"is_preprint":false},{"year":2009,"finding":"The C-terminal domain of Cdt1 adopts a winged-helix fold; the winged helix is also conserved in the middle domain; structural analysis defines a binding site for the MCM complex within the C-terminal winged-helix domain.","method":"X-ray crystallography; solution NMR spectroscopy; biochemical binding analysis","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus NMR, single lab but two structural methods","pmids":["19722278"],"is_preprint":false},{"year":2010,"finding":"The C-terminal domain of human Mcm6 is the Cdt1-binding domain (CBD); its NMR structure reveals a winged-helix fold; the helix-turn-helix motif of CBD mediates the Cdt1–Mcm6 interaction; charge complementarity is a key determinant for the Cdt1–Mcm2-7 interaction; disruption of the interaction in yeast is lethal and prevents MCM2 chromatin loading.","method":"NMR structure determination; site-directed mutagenesis; yeast in vivo MCM loading and viability assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure, mutagenesis validated in vivo in yeast; replicated by PMID 22140117","pmids":["20202939","22140117"],"is_preprint":false},{"year":2010,"finding":"Cdt1 induces large-scale chromatin decondensation required for MCM recruitment during G1; this requires HBO1 HAT activity and histone H4 modifications; HDAC11 inhibits Cdt1-induced chromatin unfolding and binds Cdt1 more efficiently in the presence of Geminin; the process is suppressed by Geminin.","method":"Live-cell imaging of large-scale chromatin unfolding; MCM loading assay; HBO1 and HDAC11 overexpression/knockdown","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — live imaging with functional assay but single lab, single study","pmids":["20980834"],"is_preprint":false},{"year":2011,"finding":"p97 AAA+-ATPase and its cofactor UFD1 are required for proteasome-dependent removal of ubiquitinated CDT1 (and SET8) from chromatin and their subsequent degradation, both in vivo and in Xenopus egg extracts; a genome-wide siRNA screen also identified nucleotide excision repair factors as promoting CDT1 destruction by recruiting PCNA to damaged DNA.","method":"Genome-wide siRNA screen; Xenopus egg extract in vitro chromatin extraction assay; immunoprecipitation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen plus Xenopus in vitro system, independently confirmed by PMID 21981920","pmids":["21981919","21981920"],"is_preprint":false},{"year":2011,"finding":"CDC-48/p97 and its cofactors UFD-1/NPL-4 coordinate CDT-1 degradation with release of the CDC-45/GINS complex from chromatin; C. elegans embryos lacking CDC-48 accumulate CDT-1 on mitotic chromatin and show persistent CDC-45/GINS chromatin association that is a consequence of CDT-1 stabilization.","method":"C. elegans RNAi; chromatin fractionation; Xenopus egg extract confirmation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo C. elegans genetics/RNAi plus Xenopus biochemical confirmation; two independent systems","pmids":["21981920"],"is_preprint":false},{"year":2011,"finding":"CRL4(Cdt2) uses two different ubiquitin-conjugating enzymes for different substrates: UBCH8 ubiquitylates p21 and Set8, while UBE2G1/UBE2G2 polyubiquitylate Cdt1 post-radiation.","method":"Proteomic analysis of Cdt2-associated proteins; siRNA screening; in vitro and in vivo ubiquitination assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination reconstitution, siRNA, single lab but multiple orthogonal approaches","pmids":["21628527"],"is_preprint":false},{"year":2011,"finding":"SNF2H chromatin remodeler is recruited to replication origins in a Cdt1-dependent manner; SNF2H physically interacts with Cdt1; SNF2H silencing prevents MCM loading at origins and inhibits S-phase progression.","method":"Co-immunoprecipitation; chromatin immunoprecipitation; siRNA knockdown; MCM loading assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP, ChIP, siRNA; single lab, single study","pmids":["21937426"],"is_preprint":false},{"year":2011,"finding":"JNK phosphorylates Cdt1 on threonine 29 in response to non-genotoxic stress, leading to rapid dissociation of HBO1 from replication origins and blocking initiation of DNA replication; upon stress relief, HBO1 reassociates with origins.","method":"In vitro kinase assay; chromatin immunoprecipitation; mutagenesis; siRNA","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, ChIP, mutagenesis; single lab, multiple methods","pmids":["21856198"],"is_preprint":false},{"year":2011,"finding":"Stress-activated MAP kinases p38 and JNK phosphorylate Cdt1 during G2 phase and acute stress; phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation by blocking Cdt1 binding to the CUL4 adaptor Cdt2; phosphomimetic mutations reduce Cdt1's ability to support origin licensing.","method":"In vitro kinase assay; co-immunoprecipitation; mutagenesis; cell synchronization; ubiquitination assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase, co-IP, mutagenesis, functional licensing assay; single lab multiple methods","pmids":["21930785"],"is_preprint":false},{"year":2011,"finding":"Multiple Cdt1 molecules are recruited to each origin by ORC and Cdc6 during helicase loading; disruption of this multi-Cdt1 intermediate prevents Mcm2-7 loading; the C-terminus of Cdt1 binds Mcm2-7 and is required for origin recruitment; the essential N-terminal domain of Cdt1 is required to load Mcm2-7 competent for subsequent replication initiation.","method":"In vitro helicase-loading assay with purified yeast proteins; domain mutation analysis; quantitative biochemistry","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins, mutagenesis, quantitative analysis","pmids":["22045335"],"is_preprint":false},{"year":2011,"finding":"Cdt1 is dynamically recruited to sites of DNA damage (laser nanosurgery) in G1 phase within seconds, dependent on PCNA; PCNA and Cdt2 recruitment to damage are independent of Cdt1; PCNA provides an immobile binding interface for dynamic Cdt1 interactions that precede its degradation.","method":"Laser nanosurgery; live-cell fluorescence microscopy; FRAP with reaction-diffusion modeling; siRNA knockdown","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — quantitative live imaging, FRAP with modeling, siRNA; single lab multiple methods","pmids":["21224399"],"is_preprint":false},{"year":2012,"finding":"Human Cdt1 localizes to kinetochores during mitosis through direct interaction with the loop domain of Hec1 (Ndc80 complex); G2-specific Cdt1 depletion causes unstable kinetochore-microtubule attachments and Mad1-dependent spindle-assembly checkpoint arrest; Cdt1 binding to the Hec1 loop promotes a microtubule-dependent conformational change in the Ndc80 complex.","method":"G2-specific siRNA depletion; co-immunoprecipitation; super-resolution fluorescence microscopy; kinetochore-microtubule attachment assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — G2-specific depletion with defined mitotic phenotype, co-IP, super-resolution microscopy; single lab but multiple orthogonal methods","pmids":["22581055"],"is_preprint":false},{"year":2012,"finding":"FOXO3 forms a protein complex with Cdt1 that blocks Cdt1 interaction with DDB1 and PCNA; FOXO3 depletion facilitates Cdt1 proteolysis in unperturbed cells and impairs S-phase entry.","method":"Co-immunoprecipitation; FOXO3 siRNA; S-phase entry assay; competition binding assay","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP, siRNA with S-phase phenotype; single lab","pmids":["22451935"],"is_preprint":false},{"year":2013,"finding":"Cryo-EM structure of the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate shows that ORC-Cdc6 N-terminal AAA+ domains latch onto Mcm2-7 C-terminal AAA+ domains; ORC-Cdc6 adopts a right-handed spiral with helical symmetry matching the DNA double helix, resembling the RFC clamp loader.","method":"Cryo-electron microscopy; in vitro assembly with purified components and ATPγS","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure of assembled complex with purified components","pmids":["23851460"],"is_preprint":false},{"year":2014,"finding":"SCF-FBXO31 E3 ligase interacts with Cdt1 and ubiquitylates it specifically in G2 phase, independently of the previously described S-phase degradation pathways; FBXO31 depletion stabilizes Cdt1 and causes re-replication.","method":"Co-immunoprecipitation; in vitro ubiquitylation assay; cell cycle synchronization; siRNA knockdown; re-replication assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro ubiquitylation, co-IP, cell cycle assay; single lab","pmids":["24828503"],"is_preprint":false},{"year":2015,"finding":"GRWD1 is a histone-binding protein recruited to replication origins in a CDC6- and Cdt1-dependent manner during G1; GRWD1 depletion reduces MCM loading; GRWD1 regulates chromatin openness at origin-proximate loci.","method":"Chromatin immunoprecipitation-seq; FAIRE-seq; siRNA depletion; MCM loading assay; histone-binding assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ChIP-seq, FAIRE-seq, siRNA with MCM loading readout; single lab","pmids":["25990725"],"is_preprint":false},{"year":2016,"finding":"USP37 is a deubiquitinase that interacts with Cdt1, deubiquitinates it in vivo, and stabilizes it preferentially during G1/G1-S; USP37 knockdown destabilizes Cdt1 and reduces MCM loading and DNA replication fork speed.","method":"Co-immunoprecipitation; in vivo deubiquitination assay; overexpression screen; siRNA knockdown; MCM loading assay; DNA fiber assay","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP, in vivo deubiquitination, functional assays; single lab","pmids":["27296872"],"is_preprint":false},{"year":2017,"finding":"3.9-Å cryo-EM structure of the yeast OCCM on DNA shows that Cdt1 has a three-domain configuration embracing Mcm2, Mcm4, and Mcm6 (nearly half the hexamer); the Cdt1 C-terminal domain contacts the Mcm6 WHD, which binds Orc4 WHD; flexible Mcm2-7 WHDs engage ORC-Cdc6; origin DNA is threaded through both rings.","method":"Cryo-EM at 3.9-Å resolution with in vitro assembled OCCM on DNA; domain mapping","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic cryo-EM structure of intact complex on DNA","pmids":["28191893"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM and X-ray crystallography show that Cdt1 contains two winged-helix domains in its C-terminal half and a catalytically inactive dioxygenase-related N-terminal domain; Cdt1 interacts with Mcm2, 4, and 6, destabilizes the Mcm2-5 interface, and inhibits MCM ATPase activity; Cdt1 stabilizes MCM in a left-handed spiral with the Mcm2-5 gate open, acting as a brace for DNA entry until ORC-Cdc6 triggers ATP hydrolysis, Cdt1 ejection, and ring closure.","method":"X-ray crystallography; single-particle electron microscopy; in vitro ATPase assay; MCM loading reconstitution","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, EM, in vitro enzymatic assay, mechanistic reconstitution in single study","pmids":["28643783"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM of the yeast Cdt1-MCM heptamer shows that both MCM hexamer and Cdt1-MCM heptamer form left-handed coil structures with a 10–15-Å gap between Mcm5 and Mcm2; Cdt1 wraps around N-terminal regions of Mcm2, Mcm6, and Mcm4 to stabilize the open ring complex.","method":"Cryo-EM of purified yeast Cdt1-MCM complexes","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure; independently corroborated by PMID 28191893 and 28643783","pmids":["28191894"],"is_preprint":false},{"year":2021,"finding":"SLFN11 promotes CDT1 degradation in response to replicative DNA damage by binding to DDB1 of the CUL4(CDT2) E3 ubiquitin ligase at replication forks; the C-terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation; an ATPase-dead SLFN11 mutant (E669K) found in cancer contributes to chemoresistance and retarded CDT1 degradation.","method":"Co-immunoprecipitation; siRNA knockdown; domain mutagenesis; CDT1 stability assay","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP, mutagenesis, functional CDT1 degradation assay; single lab","pmids":["33536335"],"is_preprint":false},{"year":2023,"finding":"CDT1 directly inhibits the CMG helicase at replication forks during an overlap period in early S phase when CDT1 is still present after origin firing; CDT1 suppresses CMG helicase activity, and DNA synthesis begins only after CDT1 is degraded; this separates licensing from DNA synthesis rather than strictly separating licensing from firing.","method":"Quantitative single-cell microscopy; in vitro reconstituted human DNA replication with purified CMG and CDT1; biochemical helicase assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified human CMG and CDT1, quantitative single-cell microscopy; single lab but two orthogonal methods","pmids":["36608667"],"is_preprint":false}],"current_model":"CDT1 is an essential replication licensing factor that, together with ORC, Cdc6, and MCM2-7, assembles prereplicative complexes in G1; structural studies show CDT1 embraces MCM2, MCM4, and MCM6 to hold the hexameric ring open for DNA entry and inhibit MCM ATPase until ORC-Cdc6 triggers ring closure and CDT1 ejection. After origin firing, CDT1 directly suppresses CMG helicase activity until it is degraded, thereby separating licensing from DNA synthesis. CDT1 abundance and activity are tightly restricted by multiple mechanisms: two E3 ubiquitin ligases (CRL4-DDB1(CDT2), which requires PCNA-docking via a PIP-box motif, and SCF-Skp2, which requires CDK-mediated phosphorylation at T29/Cy-motif), with additional input from APC/C(Cdh1), SCF-FBXO31, and the deubiquitinase USP37; geminin inhibits CDT1 by forming a heterohexameric complex that sterically blocks MCM access; CDKs phosphorylate CDT1 to both destabilize it via Skp2 and inhibit its intrinsic DNA-binding activity; MAP kinases (p38/JNK) phosphorylate CDT1 in G2/stress to stabilize it and block its degradation by CRL4(CDT2); acetylation by KAT2B/KAT3B and deacetylation by HDAC11 provide an additional stability switch; p97/CDC-48 ATPase extracts ubiquitinated CDT1 from chromatin for proteasomal degradation; SLFN11 promotes CDT1 degradation under replicative stress via DDB1 recruitment; and in mitosis CDT1 relocates to kinetochores via the Hec1 loop domain of the Ndc80 complex to support stable kinetochore-microtubule attachment."},"narrative":{"mechanistic_narrative":"CDT1 is an essential DNA replication licensing factor that acts in G1 to load the MCM2-7 replicative helicase onto origins, thereby coupling S phase to mitosis in the cell-division cycle [PMID:8313888, PMID:10766248, PMID:11896191]. Working together with ORC, Cdc6, and the MCM2-7 hexamer, CDT1 assembles prereplicative complexes; structural studies of the ORC-Cdc6-Cdt1-MCM2-7 (OCCM) intermediate show that CDT1 embraces MCM2, MCM4, and MCM6 (nearly half the hexamer), destabilizes the Mcm2-5 interface, and inhibits MCM ATPase to brace the ring open for DNA entry until ORC-Cdc6-triggered ATP hydrolysis drives ring closure and CDT1 ejection [PMID:28191893, PMID:28643783, PMID:28191894, PMID:23851460]. CDT1 contacts MCM through its C-terminal winged-helix domain and the Mcm6 Cdt1-binding domain, an interaction governed by charge complementarity and essential for MCM chromatin loading [PMID:19722278, PMID:20202939, PMID:22140117, PMID:18606811, PMID:18006685]. After origin firing CDT1 directly suppresses CMG helicase activity, so DNA synthesis begins only once CDT1 is degraded, separating licensing from synthesis [PMID:36608667]. To prevent re-replication, CDT1 abundance is restricted by two redundant E3 ligases: CRL4-DDB1(CDT2), which destroys CDT1 on chromatin via a PCNA-docking PIP-box, and SCF-Skp2, which acts after CDK phosphorylation at T29/the Cy-motif, with additional control by APC/C(Cdh1) [PMID:16482215, PMID:16949367, PMID:16362051, PMID:15004027, PMID:14993212, PMID:18162579]. The inhibitor geminin binds CDT1 and blocks MCM access, switching between a permissive heterotrimer and an inhibitory heterohexamer [PMID:11125146, PMID:15286659, PMID:19906994]. CDT1 activity is further tuned by stress-activated p38/JNK phosphorylation that stabilizes it against CRL4(CDT2), by KAT2B/KAT3B acetylation opposed by HDAC11, by p97/CDC-48-mediated extraction of ubiquitinated CDT1 from chromatin, and by the deubiquitinase USP37 [PMID:21930785, PMID:21856198, PMID:19276081, PMID:21981919, PMID:21981920, PMID:27296872]. Beyond licensing, CDT1 localizes to kinetochores in mitosis via the Hec1 loop of the Ndc80 complex to support stable kinetochore-microtubule attachments [PMID:22581055].","teleology":[{"year":1994,"claim":"Established CDT1 as an essential gene whose loss blocks DNA replication while leaving mitotic events intact, defining its role in the S-phase–mitosis dependency relationship.","evidence":"Cdc10-bound genomic isolation and null allele phenotyping in fission yeast","pmids":["8313888"],"confidence":"High","gaps":["Molecular function at origins not yet defined","No partners identified"]},{"year":2002,"claim":"Defined CDT1's core molecular activity: cooperating with Cdc6 to load MCM2-7 onto chromatin during pre-RC assembly, required for initiation but not elongation, across yeast and human cells.","evidence":"Re-replication and chromatin fractionation in S. pombe, conditional depletion in budding yeast, antibody microinjection in human cells","pmids":["10766248","11836525","11967159","11896191"],"confidence":"High","gaps":["Structural basis of MCM loading not resolved","Stoichiometry of CDT1 at origins unknown"]},{"year":2001,"claim":"Identified geminin as a direct CDT1 inhibitor that suppresses replication licensing, establishing the key negative regulator of CDT1 activity.","evidence":"Xenopus egg extract replication/licensing assays, co-IP, RLF-B purification, geminin immunodepletion rescue","pmids":["11125146","11175741"],"confidence":"High","gaps":["Structural mechanism of inhibition not yet known","Whether geminin blocks MCM or DNA binding unclear at this stage"]},{"year":2004,"claim":"Resolved CDT1 domain architecture and the structural basis of geminin inhibition: a geminin coiled-coil dimer contacts both CDT1 termini and sterically blocks MCM access, with separable geminin-, MCM6-, and DNA-binding regions.","evidence":"Yeast two-hybrid/domain mapping in mouse Cdt1; crystal structures of geminin–Cdt1 and geminin with mutagenesis and replication assays","pmids":["12192004","15286659","15260975"],"confidence":"High","gaps":["Quaternary heterohexamer switch not yet appreciated","MCM-bound CDT1 structure absent"]},{"year":2006,"claim":"Delineated the dual proteolytic control of CDT1: CRL4-DDB1(CDT2) recognizing the PCNA-docking N-terminus and SCF-Skp2 acting after CDK phosphorylation, with APC/C(Cdh1) as a third ligase, preventing re-replication.","evidence":"Phosphorylation/kinase assays, PIP-box and destruction-box mutagenesis, in vivo/in vitro ubiquitination, co-silencing of Cul4/DDB1/Cdt2/Skp2, Xenopus extracts, zebrafish epistasis","pmids":["12840033","14578910","15448697","15598982","15004027","14993212","16362051","16482215","16949367","16861906","17085480","18162579"],"confidence":"High","gaps":["Quantitative contribution of each ligase across cell-cycle phases not fully partitioned","Conjugating-enzyme specificity only partly mapped"]},{"year":2007,"claim":"Established that dynamic CDT1–ORC and CDT1–chromatin associations enable iterative MCM loading and that CDT1 recruits chromatin remodeling/acetylation activity to origins.","evidence":"In vitro helicase loading with Orc6-Cdt1 fusion, FRAP live-cell imaging, co-IP with HBO1/SNF2H/GRWD1, ChIP, MCM loading assays","pmids":["18006685","17318181","18832067","21937426","25990725"],"confidence":"High","gaps":["In vivo necessity of chromatin remodelers for licensing varies between studies","Some remodeler links rest on single-lab data"]},{"year":2009,"claim":"Refined regulation of CDT1 by acetylation (KAT2B/KAT3B vs HDAC11) and by a geminin quaternary-state switch (permissive heterotrimer vs inhibitory heterohexamer) controlling licensing competence.","evidence":"In vitro acetylation/deacetylation and ubiquitylation assays; crystallography and SAXS of Cdt1–geminin complexes with functional assays","pmids":["19276081","19906994","20980834"],"confidence":"High","gaps":["Acetylation switch not independently replicated","In vivo prevalence of heterohexamer vs heterotrimer not quantified"]},{"year":2011,"claim":"Showed stress-kinase and p97-based control: p38/JNK phosphorylation at T29 stabilizes CDT1 by blocking Cdt2 binding while blocking origin firing, and p97/CDC-48-UFD1/NPL4 extracts ubiquitinated CDT1 from chromatin for degradation, coordinating helicase release.","evidence":"In vitro kinase assays, ChIP, mutagenesis, genome-wide siRNA screen, Xenopus chromatin extraction, C. elegans RNAi","pmids":["21856198","21930785","21981919","21981920"],"confidence":"High","gaps":["Physiological stresses engaging this branch incompletely defined","Cross-talk with the CRL4(CDT2) pathway not fully integrated"]},{"year":2012,"claim":"Revealed a non-licensing mitotic role: CDT1 binds the Hec1 loop of the Ndc80 complex at kinetochores to stabilize kinetochore-microtubule attachments and satisfy the spindle-assembly checkpoint.","evidence":"G2-specific siRNA depletion, co-IP, super-resolution microscopy, kinetochore-microtubule attachment assays","pmids":["22581055"],"confidence":"High","gaps":["How licensing and kinetochore functions are temporally separated unclear","Regulation of CDT1 at kinetochores not defined"]},{"year":2017,"claim":"Provided the near-atomic mechanism of licensing: CDT1 wraps MCM2/4/6, holds the Mcm2-5 gate open, inhibits MCM ATPase, and is ejected upon ORC-Cdc6-driven ATP hydrolysis and ring closure.","evidence":"Cryo-EM of OCCM on DNA at 3.9 Å, crystallography/EM with in vitro ATPase and MCM loading reconstitution","pmids":["28191893","28191894","28643783","23851460"],"confidence":"High","gaps":["Dynamics of the loading-to-firing transition partially inferred","Mammalian OCCM structural detail less complete than yeast"]},{"year":2023,"claim":"Demonstrated that CDT1 directly inhibits the CMG helicase after origin firing, so DNA synthesis begins only upon CDT1 degradation—separating licensing from synthesis rather than strictly from firing.","evidence":"In vitro reconstitution with purified human CMG and CDT1, biochemical helicase assays, quantitative single-cell microscopy","pmids":["36608667"],"confidence":"High","gaps":["Structural basis of CDT1–CMG inhibition not resolved","How this overlap window is regulated in vivo unclear"]},{"year":null,"claim":"How the many post-translational inputs (ubiquitination, phosphorylation, acetylation, deubiquitination, stress kinases) are integrated quantitatively to set CDT1 levels through the cycle, and the structural basis of CDT1's direct CMG inhibition, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified quantitative model of competing ligases and stabilizers","No structure of the CDT1–CMG inhibitory complex","Coordination of mitotic kinetochore role with licensing unaddressed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,31,52,55]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,26,43,51]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6,15]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,4,5]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[13,19,27,37]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[45]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[1,7,43,51]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,45]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[21,22,37]}],"complexes":["pre-replicative complex (ORC-Cdc6-Cdt1-MCM2-7 / OCCM)","Cdt1-geminin heterohexamer","Cdt1-MCM2-7 (Cdt1-MCM heptamer)","CRL4-DDB1(CDT2) E3 ligase substrate"],"partners":["MCM6","MCM2","GEMININ","CDC6","ORC6","DDB1","SKP2","PCNA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H211","full_name":"DNA replication factor Cdt1","aliases":["Double parked homolog","DUP"],"length_aa":546,"mass_kda":60.4,"function":"Required for both DNA replication and mitosis (PubMed:11125146, PubMed:14993212, PubMed:21856198, PubMed:22581055, PubMed:26842564). DNA replication licensing factor, required for pre-replication complex assembly. Cooperates with CDC6 and the origin recognition complex (ORC) during G1 phase of the cell cycle to promote the loading of the mini-chromosome maintenance (MCM) complex onto DNA to generate pre-replication complexes (pre-RC) (PubMed:14672932). Required also for mitosis by promoting stable kinetochore-microtubule attachments (PubMed:22581055). Potential oncogene (By similarity)","subcellular_location":"Nucleus; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q9H211/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CDT1","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/CDT1","total_profiled":1310},"omim":[{"mim_id":"620295","title":"DDB1- AND CUL4-ASSOCIATED FACTOR 10; DCAF10","url":"https://www.omim.org/entry/620295"},{"mim_id":"616396","title":"SCM POLYCOMB GROUP PROTEIN HOMOLOG 1; SCMH1","url":"https://www.omim.org/entry/616396"},{"mim_id":"616372","title":"DDB1- AND CUL4-ASSOCIATED FACTOR 4; DCAF4","url":"https://www.omim.org/entry/616372"},{"mim_id":"615167","title":"LEUCINE-RICH REPEATS- AND WD REPEAT DOMAIN-CONTAINING PROTEIN 1; LRWD1","url":"https://www.omim.org/entry/615167"},{"mim_id":"614086","title":"MULTICILIATE DIFFERENTIATION AND DNA SYNTHESIS-ASSOCIATED CELL CYCLE PROTEIN; MCIDAS","url":"https://www.omim.org/entry/614086"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":36.1}],"url":"https://www.proteinatlas.org/search/CDT1"},"hgnc":{"alias_symbol":["DUP","RIS2"],"prev_symbol":[]},"alphafold":{"accession":"Q9H211","domains":[{"cath_id":"-","chopping":"172-277_285-340","consensus_level":"high","plddt":90.4178,"start":172,"end":340},{"cath_id":"1.10.10.1420","chopping":"442-546","consensus_level":"high","plddt":88.0071,"start":442,"end":546}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H211","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H211-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H211-F1-predicted_aligned_error_v6.png","plddt_mean":73.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CDT1","jax_strain_url":"https://www.jax.org/strain/search?query=CDT1"},"sequence":{"accession":"Q9H211","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H211.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H211/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H211"}},"corpus_meta":[{"pmid":"11125146","id":"PMC_11125146","title":"Inhibition 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genomic sequences; null allele analysis showing DNA replication defect without mitotic block\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null allele with defined replication phenotype, replicated across subsequent studies in multiple organisms\",\n      \"pmids\": [\"8313888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Fission yeast Cdt1 is located in the nucleus, cooperates with Cdc18/Cdc6 to load the MCM protein Cdc21 onto chromatin at the end of mitosis, and both factors are required to initiate DNA replication; overexpression of both together drives extensive re-replication.\",\n      \"method\": \"Re-replication assay in S. pombe; chromatin fractionation; nuclear localization by microscopy; genetic epistasis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (chromatin fractionation, re-replication assay, genetic cooperation), replicated in subsequent studies\",\n      \"pmids\": [\"10766248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Geminin binds tightly to Cdt1, and excess Cdt1 reverses geminin-mediated inhibition of DNA replication in cell-free Xenopus extracts, establishing that geminin inhibits replication by targeting Cdt1.\",\n      \"method\": \"Cell-free Xenopus egg extract DNA replication assay; co-immunoprecipitation; rescue by excess Cdt1\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro replication assay with direct rescue experiment; independently replicated by multiple labs\",\n      \"pmids\": [\"11125146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RLF-B (replication licensing factor B) was purified and shown to correspond to Cdt1; geminin inhibits RLF-B/Cdt1 activity, and immunodepletion of geminin from metaphase Xenopus extracts allows assembly of licensed replication origins.\",\n      \"method\": \"Protein purification from Xenopus egg extracts; immunodepletion; replication licensing assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical purification of activity, immunodepletion rescue, replicated independently\",\n      \"pmids\": [\"11175741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Human Cdt1 (hCdt1) is a nuclear protein present only in G1; after S-phase onset its levels decrease via proteasomal proteolysis (not transcriptional changes), and hCdt1 can associate with geminin.\",\n      \"method\": \"Cell cycle synchronization; immunoblotting; proteasome inhibitor stabilization; co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple methods (synchronization, proteasome inhibition, co-IP); replicated extensively\",\n      \"pmids\": [\"11555648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Budding yeast Cdt1 (Tah11/Sid2) is required for pre-RC assembly; depletion blocks Mcm2 chromatin loading and replication initiation but not elongation; Cdt1 accumulates in the nucleus during G1 and is excluded later by CDKs, and its nuclear accumulation is interdependent with Mcm2-7.\",\n      \"method\": \"Conditional depletion; chromatin fractionation; co-immunoprecipitation; cell cycle analysis\",\n      \"journal\": \"Nature cell biology / Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independent papers (PMID 11836525 and 11967159) with depletion, chromatin fractionation, co-IP\",\n      \"pmids\": [\"11836525\", \"11967159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Mouse Cdt1 binds geminin through its central region (aa 177–380) and MCM6 through its C-terminal region (aa 407–477); Cdt1 also has intrinsic sequence-, strand-, and conformation-independent DNA binding activity that overlaps the geminin binding domain and is inhibited by geminin.\",\n      \"method\": \"Yeast two-hybrid; in vitro binding assays with purified proteins; domain deletion mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — purified proteins, domain mapping, in vitro binding; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12192004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human Cdt1 is essential for DNA replication in intact human cells; it functions during the pre-RC formation window and is required for loading MCM proteins onto chromatin.\",\n      \"method\": \"Antibody microinjection into human cells; MCM chromatin loading assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct loss-of-function by antibody microinjection with defined MCM loading readout; replicated by subsequent studies\",\n      \"pmids\": [\"11896191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The SCF(Skp2) complex ubiquitinates Cdt1 in a phosphorylation-dependent manner; Skp2 specifically interacts with Cdt1, and downregulation of Skp2 or disruption of the Cdt1–Skp2 interaction stabilizes and accumulates Cdt1.\",\n      \"method\": \"Co-immunoprecipitation; in vitro and in vivo ubiquitination assay; siRNA knockdown; dominant-negative disruption\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination reconstitution, in vivo co-IP, KD phenotype; replicated by multiple labs\",\n      \"pmids\": [\"12840033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain; geminin inhibits Cdt1 by blocking its binding to both Mcm2 and Cdc6; the Cdt1–Mcm2 association is regulated by cell growth and induced by Cdc6 expression.\",\n      \"method\": \"Co-immunoprecipitation; ectopic expression in quiescent cells; domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP, ectopic expression, single lab but multiple approaches\",\n      \"pmids\": [\"14672932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CDT1 is rapidly degraded after UV or gamma-irradiation via the CUL4-ROC1 E3 ubiquitin ligase and the COP9 signalosome; loss of either complex completely suppresses CDT1 proteolysis; CDT1 is specifically polyubiquitinated by CUL4 complexes.\",\n      \"method\": \"siRNA knockdown of CUL4, ROC1, CSN components; in vivo ubiquitination assay; irradiation-induced degradation assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockdown with defined proteolysis phenotype, in vivo ubiquitination, replicated by multiple labs\",\n      \"pmids\": [\"14578910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Crystal structure of the mouse geminin–Cdt1 complex reveals that a coiled-coil dimer of geminin interacts with both N-terminal and C-terminal portions of Cdt1; the primary interface involves steric complementarity with Pro181, Ala182, Tyr183, Phe186, and Leu189 of Cdt1, and the C-terminal region of geminin sterically blocks MCM access to Cdt1.\",\n      \"method\": \"X-ray crystallography; mutagenesis; biochemical binding assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis validation\",\n      \"pmids\": [\"15286659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"DDB1 associates stoichiometrically with CUL4A in vivo; recombinant DDB1 and CDT1 bind directly in vitro; DDB1 bridges CDT1 to CUL4A in vivo; silencing DDB1 prevents UV-induced CDT1 degradation and CUL4A-mediated CDT1 ubiquitination in vitro, establishing DDB1 as the substrate adaptor for CUL4A-mediated CDT1 ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation; in vitro direct binding with purified recombinant proteins; in vitro ubiquitination assay; siRNA knockdown\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins plus in vivo co-IP and knockdown; replicated by multiple labs\",\n      \"pmids\": [\"15448697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the first round of DNA replication in Xenopus egg extracts; degradation requires Cdk2/Cyclin E, Cdc45, RPA, and polymerase alpha (i.e., replication initiation); Cdt1 is ubiquitinated on chromatin.\",\n      \"method\": \"Xenopus egg extract replication assay; immunodepletion of replication factors; ubiquitination assay on chromatin\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cell-free reconstitution with defined factor depletions, chromatin ubiquitination assay\",\n      \"pmids\": [\"15598982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Cdt1 is phosphorylated by Cdk2 and Cdk4; phosphorylation at a cyclin-binding motif promotes Skp2 binding and subsequent SCF(Skp2)-mediated degradation; CDK inhibitors (p21, p27) suppress Cdt1 phosphorylation and stabilize it; a quadruple Cdk-binding site mutant fails to bind Skp2 and is more stable.\",\n      \"method\": \"In vitro kinase assay; co-immunoprecipitation; site-directed mutagenesis; overexpression of CDK inhibitors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay, mutagenesis, in vivo co-IP; replicated by parallel study PMID 14993212\",\n      \"pmids\": [\"15004027\", \"14993212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cyclin A-dependent CDK phosphorylation of Cdt1 (dependent on its cyclin-binding motif) promotes Skp2 binding and degradation, and also inhibits Cdt1 DNA-binding activity in vitro; however, geminin binding to Cdt1 is not affected by phosphorylation; inactivation of Cdk1 in G2/M leads to Cdt1 dephosphorylation and re-binding to chromatin.\",\n      \"method\": \"In vitro kinase assay; co-immunoprecipitation; in vitro DNA binding assay; synchronization of FT210 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro assays with mutagenesis, in vivo cell cycle validation, single lab\",\n      \"pmids\": [\"14993212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Geminin forms a parallel coiled-coil homodimer; point mutations disrupting dimerization abolish Cdt1 interaction and replication inhibition; a bipartite interface exists—an array of glutamic acid residues on the coiled-coil surface contacts positive charges in the middle of Cdt1, and an adjoining region independently contacts the N-terminal 100 residues of Cdt1; both interactions are essential for replication inhibition.\",\n      \"method\": \"X-ray crystallography of geminin; mutagenesis; in vitro replication assay; binding assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure plus mutagenesis plus functional replication assay\",\n      \"pmids\": [\"15260975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Geminin protects CDT1 from proteasome-mediated degradation (by inhibiting its ubiquitination) during S phase and allows CDT1 accumulation during mitosis, establishing a positive role for Geminin in CDT1 stability; CDK1 inhibition in mitosis, not Geminin depletion, is sufficient for premature pre-RC formation in human cells.\",\n      \"method\": \"siRNA depletion; ubiquitination assay; cell synchronization; immunoblotting\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with ubiquitination assay, single lab with two orthogonal methods\",\n      \"pmids\": [\"15257290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cdt1 proteolysis during S phase in mammalian cells occurs through the N-terminal region (not through geminin binding), is ubiquitin-dependent, and occurs independently of geminin; the N terminus is required for nuclear localization and cyclin A association but is dispensable for geminin association; N-terminus-truncated Cdt1 is stable in S phase.\",\n      \"method\": \"In vivo ubiquitination assay; siRNA for geminin; temperature-sensitive cell lines; domain truncation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ubiquitination assay, siRNA, domain truncation), replicated\",\n      \"pmids\": [\"15138268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Replication-dependent proteolysis of Cdt1 in Xenopus egg extracts requires Cdt1 interaction with PCNA through a consensus PIP-box motif; removal of PCNA inhibits Cdt1 destruction; DDB1 (Cul4 E3 component) chromatin loading depends on Cdt1–PCNA binding; mutation of the PIP-box yields stabilized Cdt1 that induces re-replication.\",\n      \"method\": \"Xenopus egg extract replication assay; PIP-box mutagenesis; PCNA depletion; chromatin fractionation; re-replication assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cell-free reconstitution, mutagenesis, depletion; replicated by multiple labs\",\n      \"pmids\": [\"16362051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for Skp2 interaction; however, T29 and the Cy motif are not necessary for S-phase proteolysis—a Skp2-independent pathway requiring the first 32 residues of Cdt1 is critical for degradation during S phase and is necessary for normal S-phase progression.\",\n      \"method\": \"Phosphopeptide mapping; mutagenesis; co-immunoprecipitation; cell cycle analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phosphopeptide mapping, mutagenesis, domain localization; single lab multiple methods\",\n      \"pmids\": [\"15855168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Two distinct E3 ubiquitin ligases regulate human Cdt1 proteolysis: DDB1-Cul4 recognizes the first ~10 amino acids (six conserved residues required for PCNA binding); SCF-Skp2 recognizes a Cy-motif region after cyclin E/A-CDK phosphorylation. Both are redundantly required in S-G2; PCNA is essential for Cul4- but not Skp2-directed degradation.\",\n      \"method\": \"Domain mutagenesis; siRNA co-silencing of Skp2 and Cul4; in vivo ubiquitination assay; PCNA binding assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis, co-silencing, in vivo ubiquitination; replicated by parallel studies\",\n      \"pmids\": [\"16482215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DCAF2/Cdt2 (a WD40 protein) functions as the substrate receptor of the CUL4-DDB1 E3 ligase to destroy Cdt1 in S phase and after DNA damage in both Xenopus and human cells; depletion of Cdt2 causes rereplication and checkpoint activation; in Xenopus, Cdt2 is recruited to replication forks via Cdt1 and PCNA, where Cdt1 ubiquitylation occurs.\",\n      \"method\": \"Identification by affinity chromatography/MS; siRNA depletion; cell cycle analysis; Xenopus egg extract assay; chromatin fractionation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS identification, depletion phenotype, Xenopus biochemistry; replicated by multiple labs\",\n      \"pmids\": [\"16949367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal region of Cdt1 contains a PCNA interaction motif required for N-terminal ubiquitination by Cul4/DDB1 after DNA damage and in S phase; the CDK2/Skp2 pathway is not essential for S-phase degradation.\",\n      \"method\": \"Domain mutagenesis; siRNA knockdown of Cul4, DDB1; in vivo ubiquitination assay; PCNA binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with in vivo ubiquitination, parallel independent confirmation (PMID 16482215, 16362051)\",\n      \"pmids\": [\"16407252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"L2DTL/CDT2 associates with CUL4, DDB1, and PCNA; loss of L2DTL suppresses CDT1 proteolysis after DNA damage; PCNA loss also suppresses CDT1 proteolysis; inactivation of L2DTL dissociates DDB1 from the CUL4 complex in vivo.\",\n      \"method\": \"Anti-CUL4 affinity chromatography/MS; co-immunoprecipitation; siRNA knockdown; PCNA silencing\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS identification, co-IP, siRNA; replicated by PMID 16949367 and others\",\n      \"pmids\": [\"16861906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DTL/CDT2 is required for CDT1 downregulation in unperturbed cells and following DNA damage via the CUL4-DDB1 complex; cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels (genetic epistasis).\",\n      \"method\": \"Zebrafish genetic screen; siRNA in human cells; genetic epistasis (Cdt1 knockdown suppresses Dtl phenotype); co-immunoprecipitation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis plus siRNA plus co-IP across two organisms\",\n      \"pmids\": [\"17085480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Orc6 directly binds Cdt1 through two regions; ORC lacking Orc6 fails to interact with Cdt1 and cannot load Mcm2-7 onto origin DNA; a fusion protein linking Cdt1 to the Orc6-CTD restores single-round Mcm2-7 loading, showing that dynamic Cdt1–ORC association is required for multiple rounds of loading.\",\n      \"method\": \"In vitro helicase-loading assay with purified proteins; direct binding assay; Orc6-Cdt1 fusion protein reconstitution\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components, fusion protein rescue, domain mapping\",\n      \"pmids\": [\"18006685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Cdt1 associates dynamically with chromatin throughout G1 (shown by FRAP); Cdt1 simultaneously binds geminin and chromatin in vivo (contrary to in vitro data) and recruits geminin onto chromatin; chromatin-binding and geminin-binding domains are separable.\",\n      \"method\": \"Quantitative fluorescence microscopy (FRAP); domain mapping; live-cell imaging in human cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRAP with functional domain analysis, single lab but multiple quantitative methods\",\n      \"pmids\": [\"17318181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"APC/C(Cdh1) is a third ubiquitin ligase that controls Cdt1 levels via three destruction boxes in the Cdt1 N-terminus; elimination of these boxes causes strong rereplication and chromosomal damage; Cdt1 also interacts with SNF2H, topoisomerases I and IIα, and GRWD1/WDR28.\",\n      \"method\": \"Cdt1 affinity chromatography/MS; co-immunoprecipitation; destruction box mutagenesis; rereplication assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with rereplication readout, co-IP, MS; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18162579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HBO1 histone acetylase directly interacts with Cdt1; HBO1 associates with replication origins during G1 in a Cdt1-dependent (but Geminin-independent) manner; HBO1 enhances Cdt1-dependent rereplication, establishing HBO1 as a coactivator of the Cdt1 licensing factor at origins.\",\n      \"method\": \"Co-immunoprecipitation; chromatin immunoprecipitation; rereplication assay; siRNA depletion\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ChIP, rereplication assay; single lab, multiple methods\",\n      \"pmids\": [\"18832067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MCM9 forms a stable complex with Cdt1 and is required for Mcm2-7 chromatin loading; MCM9 prevents excess geminin on chromatin during licensing, functioning as an activating linker between Cdt1 and Mcm2-7.\",\n      \"method\": \"Co-immunoprecipitation; immunodepletion in Xenopus extracts; chromatin fractionation; DNA replication assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, Xenopus depletion/reconstitution, chromatin fractionation; single lab multiple methods\",\n      \"pmids\": [\"18657502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Cdt1 forms a stable complex with Mcm4/6/7 and Mcm2/3/4/5/6/7 via interaction with Mcm2 and Mcm4/6; Cdt1 stimulates DNA binding and helicase activity of Mcm4/6/7 in vitro; a Cdt1 mutant failing to stimulate helicase activity shows reduced MCM interaction and is deficient in forming the high-MW MCM-Cdt1 assembly.\",\n      \"method\": \"Glycerol gradient fractionation with purified proteins; in vitro DNA binding and helicase assay; mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins, helicase assay, mutagenesis; single lab\",\n      \"pmids\": [\"18606811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cdt1 undergoes acetylation at its N terminus by KAT2B and KAT3B; HDAC11 directly binds and deacetylates Cdt1; acetylation protects Cdt1 from ubiquitylation and proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation; in vitro acetylation assay; HDAC11 binding assay; ubiquitylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro acetylation reconstitution with single lab, no independent replication identified\",\n      \"pmids\": [\"19276081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The human Cdt1–Geminin complex can exist as a 'permissive' heterotrimer or an 'inhibitory' heterohexamer; specific Cdt1 residues buried in the heterohexamer are important for licensing; the quaternary-state switch between forms represents a molecular switch between licensing-competent and -defective states.\",\n      \"method\": \"X-ray crystallography; small-angle X-ray scattering; functional assays in Xenopus and mammalian cells; mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, SAXS, mutagenesis, functional assays in two systems\",\n      \"pmids\": [\"19906994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The C-terminal domain of Cdt1 adopts a winged-helix fold; the winged helix is also conserved in the middle domain; structural analysis defines a binding site for the MCM complex within the C-terminal winged-helix domain.\",\n      \"method\": \"X-ray crystallography; solution NMR spectroscopy; biochemical binding analysis\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus NMR, single lab but two structural methods\",\n      \"pmids\": [\"19722278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The C-terminal domain of human Mcm6 is the Cdt1-binding domain (CBD); its NMR structure reveals a winged-helix fold; the helix-turn-helix motif of CBD mediates the Cdt1–Mcm6 interaction; charge complementarity is a key determinant for the Cdt1–Mcm2-7 interaction; disruption of the interaction in yeast is lethal and prevents MCM2 chromatin loading.\",\n      \"method\": \"NMR structure determination; site-directed mutagenesis; yeast in vivo MCM loading and viability assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure, mutagenesis validated in vivo in yeast; replicated by PMID 22140117\",\n      \"pmids\": [\"20202939\", \"22140117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Cdt1 induces large-scale chromatin decondensation required for MCM recruitment during G1; this requires HBO1 HAT activity and histone H4 modifications; HDAC11 inhibits Cdt1-induced chromatin unfolding and binds Cdt1 more efficiently in the presence of Geminin; the process is suppressed by Geminin.\",\n      \"method\": \"Live-cell imaging of large-scale chromatin unfolding; MCM loading assay; HBO1 and HDAC11 overexpression/knockdown\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — live imaging with functional assay but single lab, single study\",\n      \"pmids\": [\"20980834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"p97 AAA+-ATPase and its cofactor UFD1 are required for proteasome-dependent removal of ubiquitinated CDT1 (and SET8) from chromatin and their subsequent degradation, both in vivo and in Xenopus egg extracts; a genome-wide siRNA screen also identified nucleotide excision repair factors as promoting CDT1 destruction by recruiting PCNA to damaged DNA.\",\n      \"method\": \"Genome-wide siRNA screen; Xenopus egg extract in vitro chromatin extraction assay; immunoprecipitation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen plus Xenopus in vitro system, independently confirmed by PMID 21981920\",\n      \"pmids\": [\"21981919\", \"21981920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CDC-48/p97 and its cofactors UFD-1/NPL-4 coordinate CDT-1 degradation with release of the CDC-45/GINS complex from chromatin; C. elegans embryos lacking CDC-48 accumulate CDT-1 on mitotic chromatin and show persistent CDC-45/GINS chromatin association that is a consequence of CDT-1 stabilization.\",\n      \"method\": \"C. elegans RNAi; chromatin fractionation; Xenopus egg extract confirmation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo C. elegans genetics/RNAi plus Xenopus biochemical confirmation; two independent systems\",\n      \"pmids\": [\"21981920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CRL4(Cdt2) uses two different ubiquitin-conjugating enzymes for different substrates: UBCH8 ubiquitylates p21 and Set8, while UBE2G1/UBE2G2 polyubiquitylate Cdt1 post-radiation.\",\n      \"method\": \"Proteomic analysis of Cdt2-associated proteins; siRNA screening; in vitro and in vivo ubiquitination assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination reconstitution, siRNA, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"21628527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SNF2H chromatin remodeler is recruited to replication origins in a Cdt1-dependent manner; SNF2H physically interacts with Cdt1; SNF2H silencing prevents MCM loading at origins and inhibits S-phase progression.\",\n      \"method\": \"Co-immunoprecipitation; chromatin immunoprecipitation; siRNA knockdown; MCM loading assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP, ChIP, siRNA; single lab, single study\",\n      \"pmids\": [\"21937426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"JNK phosphorylates Cdt1 on threonine 29 in response to non-genotoxic stress, leading to rapid dissociation of HBO1 from replication origins and blocking initiation of DNA replication; upon stress relief, HBO1 reassociates with origins.\",\n      \"method\": \"In vitro kinase assay; chromatin immunoprecipitation; mutagenesis; siRNA\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, ChIP, mutagenesis; single lab, multiple methods\",\n      \"pmids\": [\"21856198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Stress-activated MAP kinases p38 and JNK phosphorylate Cdt1 during G2 phase and acute stress; phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation by blocking Cdt1 binding to the CUL4 adaptor Cdt2; phosphomimetic mutations reduce Cdt1's ability to support origin licensing.\",\n      \"method\": \"In vitro kinase assay; co-immunoprecipitation; mutagenesis; cell synchronization; ubiquitination assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase, co-IP, mutagenesis, functional licensing assay; single lab multiple methods\",\n      \"pmids\": [\"21930785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Multiple Cdt1 molecules are recruited to each origin by ORC and Cdc6 during helicase loading; disruption of this multi-Cdt1 intermediate prevents Mcm2-7 loading; the C-terminus of Cdt1 binds Mcm2-7 and is required for origin recruitment; the essential N-terminal domain of Cdt1 is required to load Mcm2-7 competent for subsequent replication initiation.\",\n      \"method\": \"In vitro helicase-loading assay with purified yeast proteins; domain mutation analysis; quantitative biochemistry\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins, mutagenesis, quantitative analysis\",\n      \"pmids\": [\"22045335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Cdt1 is dynamically recruited to sites of DNA damage (laser nanosurgery) in G1 phase within seconds, dependent on PCNA; PCNA and Cdt2 recruitment to damage are independent of Cdt1; PCNA provides an immobile binding interface for dynamic Cdt1 interactions that precede its degradation.\",\n      \"method\": \"Laser nanosurgery; live-cell fluorescence microscopy; FRAP with reaction-diffusion modeling; siRNA knockdown\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative live imaging, FRAP with modeling, siRNA; single lab multiple methods\",\n      \"pmids\": [\"21224399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human Cdt1 localizes to kinetochores during mitosis through direct interaction with the loop domain of Hec1 (Ndc80 complex); G2-specific Cdt1 depletion causes unstable kinetochore-microtubule attachments and Mad1-dependent spindle-assembly checkpoint arrest; Cdt1 binding to the Hec1 loop promotes a microtubule-dependent conformational change in the Ndc80 complex.\",\n      \"method\": \"G2-specific siRNA depletion; co-immunoprecipitation; super-resolution fluorescence microscopy; kinetochore-microtubule attachment assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — G2-specific depletion with defined mitotic phenotype, co-IP, super-resolution microscopy; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"22581055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"FOXO3 forms a protein complex with Cdt1 that blocks Cdt1 interaction with DDB1 and PCNA; FOXO3 depletion facilitates Cdt1 proteolysis in unperturbed cells and impairs S-phase entry.\",\n      \"method\": \"Co-immunoprecipitation; FOXO3 siRNA; S-phase entry assay; competition binding assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP, siRNA with S-phase phenotype; single lab\",\n      \"pmids\": [\"22451935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cryo-EM structure of the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate shows that ORC-Cdc6 N-terminal AAA+ domains latch onto Mcm2-7 C-terminal AAA+ domains; ORC-Cdc6 adopts a right-handed spiral with helical symmetry matching the DNA double helix, resembling the RFC clamp loader.\",\n      \"method\": \"Cryo-electron microscopy; in vitro assembly with purified components and ATPγS\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure of assembled complex with purified components\",\n      \"pmids\": [\"23851460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SCF-FBXO31 E3 ligase interacts with Cdt1 and ubiquitylates it specifically in G2 phase, independently of the previously described S-phase degradation pathways; FBXO31 depletion stabilizes Cdt1 and causes re-replication.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitylation assay; cell cycle synchronization; siRNA knockdown; re-replication assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro ubiquitylation, co-IP, cell cycle assay; single lab\",\n      \"pmids\": [\"24828503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GRWD1 is a histone-binding protein recruited to replication origins in a CDC6- and Cdt1-dependent manner during G1; GRWD1 depletion reduces MCM loading; GRWD1 regulates chromatin openness at origin-proximate loci.\",\n      \"method\": \"Chromatin immunoprecipitation-seq; FAIRE-seq; siRNA depletion; MCM loading assay; histone-binding assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ChIP-seq, FAIRE-seq, siRNA with MCM loading readout; single lab\",\n      \"pmids\": [\"25990725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"USP37 is a deubiquitinase that interacts with Cdt1, deubiquitinates it in vivo, and stabilizes it preferentially during G1/G1-S; USP37 knockdown destabilizes Cdt1 and reduces MCM loading and DNA replication fork speed.\",\n      \"method\": \"Co-immunoprecipitation; in vivo deubiquitination assay; overexpression screen; siRNA knockdown; MCM loading assay; DNA fiber assay\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP, in vivo deubiquitination, functional assays; single lab\",\n      \"pmids\": [\"27296872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"3.9-Å cryo-EM structure of the yeast OCCM on DNA shows that Cdt1 has a three-domain configuration embracing Mcm2, Mcm4, and Mcm6 (nearly half the hexamer); the Cdt1 C-terminal domain contacts the Mcm6 WHD, which binds Orc4 WHD; flexible Mcm2-7 WHDs engage ORC-Cdc6; origin DNA is threaded through both rings.\",\n      \"method\": \"Cryo-EM at 3.9-Å resolution with in vitro assembled OCCM on DNA; domain mapping\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic cryo-EM structure of intact complex on DNA\",\n      \"pmids\": [\"28191893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM and X-ray crystallography show that Cdt1 contains two winged-helix domains in its C-terminal half and a catalytically inactive dioxygenase-related N-terminal domain; Cdt1 interacts with Mcm2, 4, and 6, destabilizes the Mcm2-5 interface, and inhibits MCM ATPase activity; Cdt1 stabilizes MCM in a left-handed spiral with the Mcm2-5 gate open, acting as a brace for DNA entry until ORC-Cdc6 triggers ATP hydrolysis, Cdt1 ejection, and ring closure.\",\n      \"method\": \"X-ray crystallography; single-particle electron microscopy; in vitro ATPase assay; MCM loading reconstitution\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, EM, in vitro enzymatic assay, mechanistic reconstitution in single study\",\n      \"pmids\": [\"28643783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM of the yeast Cdt1-MCM heptamer shows that both MCM hexamer and Cdt1-MCM heptamer form left-handed coil structures with a 10–15-Å gap between Mcm5 and Mcm2; Cdt1 wraps around N-terminal regions of Mcm2, Mcm6, and Mcm4 to stabilize the open ring complex.\",\n      \"method\": \"Cryo-EM of purified yeast Cdt1-MCM complexes\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure; independently corroborated by PMID 28191893 and 28643783\",\n      \"pmids\": [\"28191894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SLFN11 promotes CDT1 degradation in response to replicative DNA damage by binding to DDB1 of the CUL4(CDT2) E3 ubiquitin ligase at replication forks; the C-terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation; an ATPase-dead SLFN11 mutant (E669K) found in cancer contributes to chemoresistance and retarded CDT1 degradation.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown; domain mutagenesis; CDT1 stability assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP, mutagenesis, functional CDT1 degradation assay; single lab\",\n      \"pmids\": [\"33536335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CDT1 directly inhibits the CMG helicase at replication forks during an overlap period in early S phase when CDT1 is still present after origin firing; CDT1 suppresses CMG helicase activity, and DNA synthesis begins only after CDT1 is degraded; this separates licensing from DNA synthesis rather than strictly separating licensing from firing.\",\n      \"method\": \"Quantitative single-cell microscopy; in vitro reconstituted human DNA replication with purified CMG and CDT1; biochemical helicase assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified human CMG and CDT1, quantitative single-cell microscopy; single lab but two orthogonal methods\",\n      \"pmids\": [\"36608667\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CDT1 is an essential replication licensing factor that, together with ORC, Cdc6, and MCM2-7, assembles prereplicative complexes in G1; structural studies show CDT1 embraces MCM2, MCM4, and MCM6 to hold the hexameric ring open for DNA entry and inhibit MCM ATPase until ORC-Cdc6 triggers ring closure and CDT1 ejection. After origin firing, CDT1 directly suppresses CMG helicase activity until it is degraded, thereby separating licensing from DNA synthesis. CDT1 abundance and activity are tightly restricted by multiple mechanisms: two E3 ubiquitin ligases (CRL4-DDB1(CDT2), which requires PCNA-docking via a PIP-box motif, and SCF-Skp2, which requires CDK-mediated phosphorylation at T29/Cy-motif), with additional input from APC/C(Cdh1), SCF-FBXO31, and the deubiquitinase USP37; geminin inhibits CDT1 by forming a heterohexameric complex that sterically blocks MCM access; CDKs phosphorylate CDT1 to both destabilize it via Skp2 and inhibit its intrinsic DNA-binding activity; MAP kinases (p38/JNK) phosphorylate CDT1 in G2/stress to stabilize it and block its degradation by CRL4(CDT2); acetylation by KAT2B/KAT3B and deacetylation by HDAC11 provide an additional stability switch; p97/CDC-48 ATPase extracts ubiquitinated CDT1 from chromatin for proteasomal degradation; SLFN11 promotes CDT1 degradation under replicative stress via DDB1 recruitment; and in mitosis CDT1 relocates to kinetochores via the Hec1 loop domain of the Ndc80 complex to support stable kinetochore-microtubule attachment.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CDT1 is an essential DNA replication licensing factor that acts in G1 to load the MCM2-7 replicative helicase onto origins, thereby coupling S phase to mitosis in the cell-division cycle [#0, #1, #7]. Working together with ORC, Cdc6, and the MCM2-7 hexamer, CDT1 assembles prereplicative complexes; structural studies of the ORC-Cdc6-Cdt1-MCM2-7 (OCCM) intermediate show that CDT1 embraces MCM2, MCM4, and MCM6 (nearly half the hexamer), destabilizes the Mcm2-5 interface, and inhibits MCM ATPase to brace the ring open for DNA entry until ORC-Cdc6-triggered ATP hydrolysis drives ring closure and CDT1 ejection [#51, #52, #53, #47]. CDT1 contacts MCM through its C-terminal winged-helix domain and the Mcm6 Cdt1-binding domain, an interaction governed by charge complementarity and essential for MCM chromatin loading [#34, #35, #31, #26]. After origin firing CDT1 directly suppresses CMG helicase activity, so DNA synthesis begins only once CDT1 is degraded, separating licensing from synthesis [#55]. To prevent re-replication, CDT1 abundance is restricted by two redundant E3 ligases: CRL4-DDB1(CDT2), which destroys CDT1 on chromatin via a PCNA-docking PIP-box, and SCF-Skp2, which acts after CDK phosphorylation at T29/the Cy-motif, with additional control by APC/C(Cdh1) [#21, #22, #19, #14, #28]. The inhibitor geminin binds CDT1 and blocks MCM access, switching between a permissive heterotrimer and an inhibitory heterohexamer [#2, #11, #33]. CDT1 activity is further tuned by stress-activated p38/JNK phosphorylation that stabilizes it against CRL4(CDT2), by KAT2B/KAT3B acetylation opposed by HDAC11, by p97/CDC-48-mediated extraction of ubiquitinated CDT1 from chromatin, and by the deubiquitinase USP37 [#42, #41, #32, #37, #38, #50]. Beyond licensing, CDT1 localizes to kinetochores in mitosis via the Hec1 loop of the Ndc80 complex to support stable kinetochore-microtubule attachments [#45].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established CDT1 as an essential gene whose loss blocks DNA replication while leaving mitotic events intact, defining its role in the S-phase\\u2013mitosis dependency relationship.\",\n      \"evidence\": \"Cdc10-bound genomic isolation and null allele phenotyping in fission yeast\",\n      \"pmids\": [\"8313888\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular function at origins not yet defined\", \"No partners identified\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined CDT1's core molecular activity: cooperating with Cdc6 to load MCM2-7 onto chromatin during pre-RC assembly, required for initiation but not elongation, across yeast and human cells.\",\n      \"evidence\": \"Re-replication and chromatin fractionation in S. pombe, conditional depletion in budding yeast, antibody microinjection in human cells\",\n      \"pmids\": [\"10766248\", \"11836525\", \"11967159\", \"11896191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of MCM loading not resolved\", \"Stoichiometry of CDT1 at origins unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified geminin as a direct CDT1 inhibitor that suppresses replication licensing, establishing the key negative regulator of CDT1 activity.\",\n      \"evidence\": \"Xenopus egg extract replication/licensing assays, co-IP, RLF-B purification, geminin immunodepletion rescue\",\n      \"pmids\": [\"11125146\", \"11175741\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism of inhibition not yet known\", \"Whether geminin blocks MCM or DNA binding unclear at this stage\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Resolved CDT1 domain architecture and the structural basis of geminin inhibition: a geminin coiled-coil dimer contacts both CDT1 termini and sterically blocks MCM access, with separable geminin-, MCM6-, and DNA-binding regions.\",\n      \"evidence\": \"Yeast two-hybrid/domain mapping in mouse Cdt1; crystal structures of geminin\\u2013Cdt1 and geminin with mutagenesis and replication assays\",\n      \"pmids\": [\"12192004\", \"15286659\", \"15260975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quaternary heterohexamer switch not yet appreciated\", \"MCM-bound CDT1 structure absent\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Delineated the dual proteolytic control of CDT1: CRL4-DDB1(CDT2) recognizing the PCNA-docking N-terminus and SCF-Skp2 acting after CDK phosphorylation, with APC/C(Cdh1) as a third ligase, preventing re-replication.\",\n      \"evidence\": \"Phosphorylation/kinase assays, PIP-box and destruction-box mutagenesis, in vivo/in vitro ubiquitination, co-silencing of Cul4/DDB1/Cdt2/Skp2, Xenopus extracts, zebrafish epistasis\",\n      \"pmids\": [\"12840033\", \"14578910\", \"15448697\", \"15598982\", \"15004027\", \"14993212\", \"16362051\", \"16482215\", \"16949367\", \"16861906\", \"17085480\", \"18162579\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each ligase across cell-cycle phases not fully partitioned\", \"Conjugating-enzyme specificity only partly mapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that dynamic CDT1\\u2013ORC and CDT1\\u2013chromatin associations enable iterative MCM loading and that CDT1 recruits chromatin remodeling/acetylation activity to origins.\",\n      \"evidence\": \"In vitro helicase loading with Orc6-Cdt1 fusion, FRAP live-cell imaging, co-IP with HBO1/SNF2H/GRWD1, ChIP, MCM loading assays\",\n      \"pmids\": [\"18006685\", \"17318181\", \"18832067\", \"21937426\", \"25990725\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo necessity of chromatin remodelers for licensing varies between studies\", \"Some remodeler links rest on single-lab data\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Refined regulation of CDT1 by acetylation (KAT2B/KAT3B vs HDAC11) and by a geminin quaternary-state switch (permissive heterotrimer vs inhibitory heterohexamer) controlling licensing competence.\",\n      \"evidence\": \"In vitro acetylation/deacetylation and ubiquitylation assays; crystallography and SAXS of Cdt1\\u2013geminin complexes with functional assays\",\n      \"pmids\": [\"19276081\", \"19906994\", \"20980834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Acetylation switch not independently replicated\", \"In vivo prevalence of heterohexamer vs heterotrimer not quantified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed stress-kinase and p97-based control: p38/JNK phosphorylation at T29 stabilizes CDT1 by blocking Cdt2 binding while blocking origin firing, and p97/CDC-48-UFD1/NPL4 extracts ubiquitinated CDT1 from chromatin for degradation, coordinating helicase release.\",\n      \"evidence\": \"In vitro kinase assays, ChIP, mutagenesis, genome-wide siRNA screen, Xenopus chromatin extraction, C. elegans RNAi\",\n      \"pmids\": [\"21856198\", \"21930785\", \"21981919\", \"21981920\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological stresses engaging this branch incompletely defined\", \"Cross-talk with the CRL4(CDT2) pathway not fully integrated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealed a non-licensing mitotic role: CDT1 binds the Hec1 loop of the Ndc80 complex at kinetochores to stabilize kinetochore-microtubule attachments and satisfy the spindle-assembly checkpoint.\",\n      \"evidence\": \"G2-specific siRNA depletion, co-IP, super-resolution microscopy, kinetochore-microtubule attachment assays\",\n      \"pmids\": [\"22581055\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How licensing and kinetochore functions are temporally separated unclear\", \"Regulation of CDT1 at kinetochores not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the near-atomic mechanism of licensing: CDT1 wraps MCM2/4/6, holds the Mcm2-5 gate open, inhibits MCM ATPase, and is ejected upon ORC-Cdc6-driven ATP hydrolysis and ring closure.\",\n      \"evidence\": \"Cryo-EM of OCCM on DNA at 3.9 \\u00c5, crystallography/EM with in vitro ATPase and MCM loading reconstitution\",\n      \"pmids\": [\"28191893\", \"28191894\", \"28643783\", \"23851460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of the loading-to-firing transition partially inferred\", \"Mammalian OCCM structural detail less complete than yeast\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that CDT1 directly inhibits the CMG helicase after origin firing, so DNA synthesis begins only upon CDT1 degradation\\u2014separating licensing from synthesis rather than strictly from firing.\",\n      \"evidence\": \"In vitro reconstitution with purified human CMG and CDT1, biochemical helicase assays, quantitative single-cell microscopy\",\n      \"pmids\": [\"36608667\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CDT1\\u2013CMG inhibition not resolved\", \"How this overlap window is regulated in vivo unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many post-translational inputs (ubiquitination, phosphorylation, acetylation, deubiquitination, stress kinases) are integrated quantitatively to set CDT1 levels through the cycle, and the structural basis of CDT1's direct CMG inhibition, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified quantitative model of competing ligases and stabilizers\", \"No structure of the CDT1\\u2013CMG inhibitory complex\", \"Coordination of mitotic kinetochore role with licensing unaddressed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 31, 52, 55]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 26, 43, 51]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 4, 5]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [13, 19, 27, 37]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [45]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [1, 7, 43, 51]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 45]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [21, 22, 37]}\n    ],\n    \"complexes\": [\n      \"pre-replicative complex (ORC-Cdc6-Cdt1-MCM2-7 / OCCM)\",\n      \"Cdt1-geminin heterohexamer\",\n      \"Cdt1-MCM2-7 (Cdt1-MCM heptamer)\",\n      \"CRL4-DDB1(CDT2) E3 ligase substrate\"\n    ],\n    \"partners\": [\n      \"MCM6\",\n      \"MCM2\",\n      \"geminin\",\n      \"CDC6\",\n      \"ORC6\",\n      \"DDB1\",\n      \"SKP2\",\n      \"PCNA\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}