{"gene":"CDC45","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2006,"finding":"CDC45, MCM2-7, and GINS form a stable replisome progression complex (RPC) at eukaryotic replication forks. GINS is essential for maintaining the association of CDC45 with MCM within RPCs after initiation, and RPCs also contain Mrc1, Tof1-Csm3, FACT, Ctf4, Mcm10, and topoisomerase I.","method":"Immunoaffinity purification, mass spectrometry, chromatin immunoprecipitation (budding yeast)","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-purification with MS identification, replicated by multiple labs in same year","pmids":["16531994"],"is_preprint":false},{"year":2006,"finding":"CDC45, MCM2-7, and GINS form the CMG (Cdc45/Mcm2-7/GINS) complex, which is the eukaryotic replicative DNA helicase. The purified complex from Drosophila embryo extracts has ATP-dependent DNA helicase activity. RNAi knockdown of GINS or CDC45 blocks S-phase transition.","method":"Immunoaffinity purification, in vitro helicase assay, RNAi knockdown (Drosophila)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro helicase assay with purified complex, corroborated by RNAi functional assay, replicated by multiple labs","pmids":["16798881"],"is_preprint":false},{"year":2010,"finding":"Association of CDC45 and GINS with MCM2-7 activates the helicase: ATP hydrolysis rates are elevated ~100-fold, helicase activity is robust on circular templates, and DNA affinity is improved. GINS binds specifically to MCM4. All pairwise associations among GINS, MCMs, and CDC45 are detectable but tight association requires the full CMG.","method":"Reconstitution with recombinant Drosophila proteins, ATPase assay, in vitro helicase assay, pulldown","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted CMG from recombinant components, multiple orthogonal biochemical assays, single rigorous study","pmids":["20122406"],"is_preprint":false},{"year":2011,"finding":"Cryo-EM structures of MCM2-7 and the CMG complex reveal that GINS and CDC45 bridge the Mcm2/Mcm5 gap in the helicase ring, forming a topologically closed assembly. Nucleotide binding further seals the ring, partitioning the central channel into two pores. This explains how GINS and CDC45 activate Mcm2-7 helicase.","method":"Single-particle electron microscopy, structural analysis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure of CMG with functional interpretation, landmark structural study","pmids":["21378962"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of human CDC45 at 2.1 Å confirms evolutionary relationship to bacterial RecJ nuclease (DHH family). Key features include: long-range N-C terminal DHH domain interaction blocking the DNA-binding groove, and a helical insertion poised for replisome interactions. Mutational analysis validated the mechanism of CDC45 association with the MCM ring and GINS co-activator critical for CMG assembly.","method":"X-ray crystallography (2.1 Å), structure-guided mutagenesis, EM data integration","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic resolution crystal structure with mutagenesis validation, multiple orthogonal methods in single study","pmids":["27189187"],"is_preprint":false},{"year":2000,"finding":"CDC45 is required for origin unwinding during replication initiation in Xenopus. CDC45 binds chromatin upstream of RPA and DNA polymerase alpha. When CDC45 is present but DNA pol alpha is inhibited, helicase activity becomes uncoupled, demonstrating that CDC45 drives the unwinding step.","method":"Xenopus egg extract replication assay, immunodepletion, supercoiling assay, chromatin fractionation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunodepletion in cell-free system with multiple functional readouts, independently replicated","pmids":["10882098"],"is_preprint":false},{"year":2006,"finding":"At paused replication forks in Xenopus, MCM2-7, CDC45, and GINS are enriched at the unwinding site even when polymerase is inhibited by aphidicolin, establishing these three as core components of the 'unwindosome' that separates DNA strands at the replication fork.","method":"Biotin-streptavidin fork-pausing assay, chromatin immunoprecipitation, Xenopus egg extracts","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct chromatin localization at defined fork position, multiple replication factors tested, Xenopus cell-free system","pmids":["16483939"],"is_preprint":false},{"year":2004,"finding":"MCM7 and CDC45 are required throughout replication elongation (not just initiation) in vertebrates. Antibody neutralization of CDC45 or MCM7 after significant DNA synthesis had already occurred blocked further synthesis and abolished helicase-dependent chromosome unwinding (uncoupled by aphidicolin), establishing CDC45 as a helicase co-factor essential for elongation.","method":"Xenopus egg extract, antibody neutralization, aphidicolin uncoupling assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — antibody depletion at defined cell cycle stages, multiple functional readouts, Xenopus system","pmids":["15329670"],"is_preprint":false},{"year":1998,"finding":"Xenopus CDC45 loads DNA polymerase alpha onto chromatin at replication initiation. CDC45 physically interacts with DNA polymerase alpha in egg extracts, associates with chromatin only after nuclear formation in an S-phase CDK-dependent manner, and co-localizes with pol alpha in S-phase nuclei.","method":"Xenopus egg extract, co-immunoprecipitation, chromatin fractionation, immunofluorescence","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP plus functional depletion in cell-free system, multiple orthogonal methods","pmids":["9755170"],"is_preprint":false},{"year":2000,"finding":"CDC45 is essential for the sequential chromatin loading of RPA, DNA pol alpha, and PCNA at replication initiation in Xenopus. CDC45 forms a stable complex with either MCM or DNA pol alpha on chromatin. DNA pol epsilon loading requires CDC45 but not pol alpha, suggesting a dual role in DNA unwinding and polymerase recruitment.","method":"Xenopus egg extract, immunodepletion, chromatin fractionation, co-immunoprecipitation","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunodepletion with multiple downstream readouts in cell-free system","pmids":["10886370"],"is_preprint":false},{"year":2001,"finding":"CDC45 forms a complex with Sld3 throughout the cell cycle in S. cerevisiae. Their origin associations are mutually dependent. In sld3 mutants, the Sld3-CDC45 interaction and the CDC45-MCM2 interaction are both reduced. RPA does not associate with origins in the absence of Sld3, showing that the Sld3-CDC45 complex is prerequisite for origin unwinding.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, two-hybrid, genetic analysis (budding yeast)","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus ChIP plus genetic epistasis, multiple orthogonal methods","pmids":["11296242"],"is_preprint":false},{"year":2012,"finding":"The human CMG complex (CDC45/MCM2-7/GINS) purified from baculovirus-infected Sf9 cells has DNA helicase activity that: requires forked DNA structures for maximal activity; translocates 3' to 5' on the leading strand template; unwinds up to 500 bp; and, together with DNA pol epsilon, supports leading-strand synthesis >10 kb.","method":"Baculovirus expression, in vitro helicase assay, ATPase assay, rolling circle DNA synthesis assay (human CMG)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted human CMG, multiple in vitro biochemical assays establishing directionality and leading-strand synthesis","pmids":["22474384"],"is_preprint":false},{"year":2015,"finding":"CDC45 guards the leading strand within the CMG: cross-linking studies show the leading strand contacts CDC45 when the MCM2/5 gate is open, but the lagging strand does not pass through the side channel. Mutations in the RecJ-like fold of CDC45 that ablate this leading-strand interaction diminish helicase activity.","method":"DNA-protein crosslinking, site-directed mutagenesis, in vitro helicase assay (Drosophila CMG)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural crosslinking with functional mutagenesis, multiple orthogonal methods","pmids":["25561522"],"is_preprint":false},{"year":2011,"finding":"CDC45 binds single-stranded DNA (ssDNA) with a structure similar to RecJ, demonstrating evolutionary relationship to DHH phosphoesterase family. Biochemical and SAXS data confirm only a subset of the Mn2+-coordinating residues are conserved, but the protein retains ssDNA (not dsDNA) binding activity.","method":"Recombinant human CDC45, SAXS, ssDNA binding assays, bioinformatics","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — biochemical assay plus structural SAXS, single lab, consistent with evolutionary prediction","pmids":["22147708"],"is_preprint":false},{"year":2013,"finding":"Human CDC45 binds long ssDNA (≥40 nt) and preferentially binds 3'-protruding strands, Y-shaped DNA, bubbles, and D-loops with higher affinity than short oligonucleotides. CDC45 slides on DNA with 3'-5' polarity, suggesting it acts as a molecular wedge to initiate strand displacement.","method":"Recombinant human CDC45, EMSA, AFM, SPR, SRCD, SAXS","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal in vitro binding assays, single lab","pmids":["24293646"],"is_preprint":false},{"year":2013,"finding":"CDC45 from budding yeast binds tightly to long (≥40 nt) ssDNA; 60-mer ssDNA disrupts the CDC45-MCM2-7 interaction. A CDC45 mutant unable to bind ssDNA causes helicase uncoupling from the polymerase under replication stress (hydroxyurea), with excess RPA accumulating near origins, demonstrating that CDC45-ssDNA interaction is required to stall the helicase during replication stress.","method":"Purified protein binding assays, site-directed mutagenesis, yeast genetics, chromatin immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding plus in vivo mutant phenotype, single lab","pmids":["23382391"],"is_preprint":false},{"year":2017,"finding":"Human CDC45 actively loads RPA onto nascent ssDNA in a catalytic manner. CDC45 forms a complex with RPA and stabilizes the 8-10 nt RPA binding mode; interaction requires the RPA70A subdomain. RPA dissociates when it covers a 30-mer. CDC45 facilitates ordered RPA deposition on ssDNA at the replication fork.","method":"Pull-down assay, surface plasmon resonance, real-time RPA-ssDNA binding assay (human recombinant proteins)","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with kinetic analysis, single lab, multiple orthogonal methods","pmids":["28100698"],"is_preprint":false},{"year":2012,"finding":"Checkpoint kinase Chk2 (but not Chk1) directly inhibits CMG helicase activity in vitro by phosphorylating MCM3, MCM4, and GINS subunit Psf2. Phosphatase treatment of CMG stimulates helicase activity. Ionizing radiation in Drosophila embryos causes hyperphosphorylation of Psf2 within the active helicase complex in vivo.","method":"In vitro kinase assay, helicase assay with recombinant Drosophila CMG, mass spectrometry, Drosophila embryo irradiation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with kinase, phosphorylation site mapping by MS, in vivo confirmation in embryos","pmids":["22853956"],"is_preprint":false},{"year":1997,"finding":"CDC45 functions in late G1 phase after START and prior to DNA synthesis to trigger initiation at replication origins. CDC45 and CDC7/Dbf4 kinase are mutually dependent for function; cells defective in CDC45 cannot activate prereplicative complexes.","method":"Yeast genetics, cell cycle synchronization, epistasis analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis, cell cycle timing experiments, single lab","pmids":["9356482"],"is_preprint":false},{"year":1997,"finding":"CDC45 is essential for DNA replication initiation in S. cerevisiae. It genetically interacts with MCM genes (CDC46, CDC47, CDC54) and is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. Origins fire less frequently in cdc45-1 cells, establishing CDC45 as functioning with ORC and MCM proteins in replication initiation.","method":"Yeast genetics, complementation, 2D gel origin firing analysis, synthetic lethality","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic interaction tests, direct 2D gel analysis of origin firing","pmids":["9001208"],"is_preprint":false},{"year":2006,"finding":"Cdc7-Dbf4 kinase (DDK)-dependent phosphorylation of MCM4 N-terminal residues stimulates CDC45 association with chromatin. Deletion of MCM4 N-terminal 150 aa causes growth inhibition, and combined alanine substitution/deletion of N-terminal segments of MCM2, MCM4, and MCM6 leads to non-viable phenotype, indicating redundant but essential roles for these DDK-target sites in CDC45 loading.","method":"Chromatin fractionation, phospho-specific antibodies, SDS-PAGE mobility shift, Cdc7 conditional KO mouse ES cells, siRNA, mutagenesis (mammalian cells)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-specific antibodies, genetic rescue, conditional KO confirmation, single lab","pmids":["17046832"],"is_preprint":false},{"year":2002,"finding":"Xenopus MCM10 binds chromatin after MCM2-7 but upstream of CDC45. In the absence of MCM10, CDC45 binding, RPA binding, and origin unwinding (supercoiling) are all blocked, placing MCM10 as an essential intermediate between pre-RC assembly and CDC45 loading.","method":"Xenopus egg extract, immunodepletion, chromatin fractionation, supercoiling assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunodepletion in cell-free system with multiple downstream readouts, epistasis ordering","pmids":["11864598"],"is_preprint":false},{"year":2002,"finding":"Protein phosphatase 2A (PP2A) is required for CDC45 loading onto the pre-RC in Xenopus. PP2A depletion or okadaic acid treatment abolishes CDC45 loading, origin unwinding, and downstream RPA and pol alpha loading. PP2A acts on a soluble factor (not CDC45 itself or pre-RC components) to enable CDC45 loading.","method":"Xenopus egg extract, PP2A immunodepletion, okadaic acid inhibition, chromatin fractionation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunodepletion plus chemical inhibition, epistasis ordering, single lab","pmids":["12185086"],"is_preprint":false},{"year":2002,"finding":"Xmus101 (TOPBP1 ortholog) is required for loading CDC45 onto origins in Xenopus. Xmus101 chromatin association depends on ORC but is independent of MCM2-7 and S-CDK, defining a parallel ORC-dependent pathway for CDC45 loading distinct from the MCM2-7 pathway.","method":"Xenopus egg extract, immunodepletion, chromatin fractionation, epistasis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunodepletion with clear epistasis ordering, single lab","pmids":["12438414"],"is_preprint":false},{"year":2011,"finding":"Origin association timing of CDC45 (together with Sld3 and Sld7) is the key determinant of origin firing time in budding yeast. CDC45 associates with early-firing origins in G1 in a DDK-dependent manner; increased dosage of Sld3/Sld7/CDC45 allows late origins to fire earlier, as does increased DDK dosage.","method":"Chromatin immunoprecipitation, dosage analysis, genetic overexpression (budding yeast)","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus overexpression genetics, single lab, clear functional readout","pmids":["22169533"],"is_preprint":false},{"year":2006,"finding":"In fission yeast, Sld3 is loaded at origins upstream of GINS, Cut5, and CDC45. DDK but not CDK is required for Sld3 loading, while CDC45 loading requires both kinases. GINS integrity is required for CDC45 loading but not Sld3 loading, establishing the ordered assembly: Sld3 → GINS/Cut5 → CDC45.","method":"Chromatin immunoprecipitation, temperature-sensitive mutant analysis, pull-down (fission yeast)","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP epistasis ordering with multiple mutants, single lab","pmids":["16990792"],"is_preprint":false},{"year":2009,"finding":"Assembly of the human CMG complex (CDC45-MCM2-7-GINS) in HeLa cells requires CDK activity, CDC7 kinase, and the additional proteins RecQL4, Ctf4/And-1, and Mcm10. CMG interactions are only observed after G1/S transition. Depletion of TopBP1 did not significantly affect CMG complex formation in human cells.","method":"Bimolecular fluorescence complementation (BiFC) in HeLa cells, siRNA depletion, CDK inhibitor treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BiFC in human cells plus siRNA epistasis, single lab","pmids":["19805216"],"is_preprint":false},{"year":2006,"finding":"Chk1-mediated S-phase checkpoint targets CDC45 via a Cdc25A/CDK2-independent mechanism. BPDE-induced DNA damage causes Chk1-dependent reduction of chromatin-associated CDC45 (not soluble CDC45) and disrupts the CDC45-MCM7 interaction at the beta-globin replication origin, without affecting MCM7, MCM10, or PCNA chromatin binding.","method":"Chromatin fractionation, co-immunoprecipitation, chromatin immunoprecipitation, Chk1 inhibitor UCN-01 (human cells)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP at specific origin plus co-IP plus specific inhibitor rescue, single lab","pmids":["16912045"],"is_preprint":false},{"year":2018,"finding":"CDC45 targets checkpoint kinase Rad53 to replication complexes via FHA-domain interaction with phosphorylated motifs in an intrinsically disordered loop of Cdc45. This interaction is necessary for Rad53-mediated inhibition of origin firing through Sld3, and also for stabilizing stalled forks. A CDC45 mutation found in Meier-Gorlin syndrome disrupts the Rad53 interaction.","method":"Co-immunoprecipitation, in vitro phosphorylation, genetic epistasis, budding yeast","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus genetic epistasis plus disease mutation validation, single lab","pmids":["30595439"],"is_preprint":false},{"year":2011,"finding":"GINS and Sld3 compete with each other for binding to both MCM2-7 and CDC45. Purified proteins form either a Cdc45-MCM2-7-Sld3 (CMS) or Cdc45-MCM2-7-GINS (CMG) complex with 1:1:1 stoichiometry. The data suggest GINS displaces Sld3 at the origin to activate the replication fork helicase.","method":"Purified recombinant protein binding assays, size exclusion chromatography, competition assays (budding yeast)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — reconstituted complexes from purified components, stoichiometry determination, single lab","pmids":["21362622"],"is_preprint":false},{"year":2021,"finding":"DDK regulates CMG formation via a two-stage mechanism: DDK phosphorylation of MCM2-7 N-terminal tails recruits Cdc45 and GINS to form Cdc45-tail-GINS (CtG) intermediates. Higher DDK phosphorylation increases CtG multiplicity per MCM2-7, and higher CtG numbers increase the frequency of CMG formation in a second, inefficient step.","method":"Single-molecule fluorescence microscopy, in vitro reconstitution, DDK phosphorylation assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — single-molecule resolution biochemical reconstitution, mechanistic ordering of two-stage assembly, single rigorous study","pmids":["33616038"],"is_preprint":false},{"year":2013,"finding":"Human CDC45 interacts with Claspin, RPA, and DNA polymerase delta maximally during S phase. UV-induced DNA damage reduces CDC45-Claspin complex formation without affecting CDC45-RPA interaction, and this dissociation occurs upstream of ATR activation in the S-phase checkpoint.","method":"Co-immunoprecipitation, synchronized HeLa cells, UV treatment, kinase inhibitors","journal":"The FEBS journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP approach, single lab, limited mechanistic follow-up","pmids":["23910567"],"is_preprint":false},{"year":2007,"finding":"Human CDC45 co-localizes with active replication sites during S phase and interacts with DNA polymerase delta, DNA polymerase epsilon, GINS subunit Psf2, and MCM5/7 subunits, suggesting CDC45 bridges replicative polymerases with the MCM helicase in the elongation complex.","method":"Co-immunoprecipitation, immunofluorescence co-localization (human cells)","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus co-localization, single lab, no functional manipulation","pmids":["17573775"],"is_preprint":false},{"year":1999,"finding":"Human CDC45 directly binds hMCM7 and the p70 subunit of DNA polymerase alpha in vitro, supporting a role as molecular tether for loading pol alpha onto the replication complex via MCM7.","method":"In vitro binding assay (direct interaction), pull-down (human proteins)","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct in vitro binding with recombinant proteins, single lab, two interaction partners characterized","pmids":["10518787"],"is_preprint":false},{"year":1998,"finding":"Human CDC45 (CDC45L) co-immunoprecipitates with human ORC2 from cell extracts; the protein associates with the nuclear fraction in G1 but this association becomes labile as S phase progresses, consistent with a role in replication initiation.","method":"Co-immunoprecipitation, subcellular fractionation (human cells)","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP, single lab, no functional manipulation","pmids":["9660782"],"is_preprint":false},{"year":2005,"finding":"Targeting CDC45 to specific chromosomal sites in mammalian cells induces large-scale chromatin decondensation correlated with histone H1 phosphorylation. CDC45 recruits CDK2 to these sites; CDK2 activity is required for decondensation. CDC45, CDK2, cyclin A, and phospho-H1 physically interact and associate with chromatin during S phase.","method":"Lac-repressor chromatin targeting, immunofluorescence, CDK2 inhibitors, co-immunoprecipitation (mammalian cells)","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct chromatin targeting with functional readout, co-IP, inhibitor validation, single lab","pmids":["15753125"],"is_preprint":false},{"year":2008,"finding":"Human TopBP1 directly interacts with CDC45 in vitro and in vivo, with this interaction occurring exclusively at the G1/S boundary. The first and second BRCT domains of TopBP1 mediate binding to CDC45, and overexpression of the sixth BRCT domain reduces CDC45 chromatin loading.","method":"GST pull-down, co-immunoprecipitation, deletion mutant analysis, yeast/mammalian one-hybrid, chromatin fractionation","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding (GST pull-down) plus co-IP plus functional chromatin loading readout, single lab","pmids":["17887956"],"is_preprint":false},{"year":2010,"finding":"DUE-B (DNA unwinding element binding protein) interacts with CDC45 and TopBP1 in cell extracts and baculovirus-expressed proteins. DUE-B and CDC45 co-localize at active replication origins. DUE-B immunodepletion in Xenopus egg extracts blocks replication and CDC45 (and a fraction of TopBP1) loading onto chromatin.","method":"Co-immunoprecipitation, baculovirus co-expression, chromatin immunoprecipitation, Xenopus immunodepletion","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus Xenopus depletion with functional readout, single lab","pmids":["20065034"],"is_preprint":false},{"year":2013,"finding":"Human Ctf4 (hCtf4) forms a complex with the CMG helicase in vitro (purified proteins), in Sf9 cells, and from HeLa chromatin. hCtf4 is a homodimer that acts as a platform linking pol alpha to CMG. The hCtf4-CMG complex retains helicase activity with greater salt resistance than CMG alone. Stability of hCtf4-CMG depends on interactions with multiple CMG components.","method":"In vitro binding with purified proteins, co-infection Sf9 cells, HeLa chromatin immunoprecipitation, helicase assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted from purified proteins plus in cellulo validation plus functional helicase assay, multiple orthogonal methods","pmids":["24255107"],"is_preprint":false},{"year":2001,"finding":"Fission yeast Sna41 (CDC45 ortholog) facilitates loading of DNA pol alpha onto MCM proteins in vivo. Sna41 interacts with pol alpha throughout the cell cycle and with Mcm6 in chromatin fractions at G1-S. In a sna41 initiation-defective mutant, pol alpha does not interact with MCM6, establishing CDC45 as essential for pol alpha-MCM association.","method":"In vivo tagged protein co-immunoprecipitation, chromatin fractionation, fission yeast genetics","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo tagged proteins, chromatin fractionation, conditional mutant epistasis, single lab","pmids":["11344166"],"is_preprint":false},{"year":2003,"finding":"Following replication initiation in Xenopus, CDC45 and all six MCM subunits form a tight complex on chromatin in a CDK-dependent manner. This MCM-CDC45 chromatin complex has DNA helicase activity, which requires both CDK activity and CDC45, providing direct evidence for CDC45 as a helicase co-factor in vivo.","method":"Xenopus egg extract, denaturing immunoprecipitation, chromatin immunoprecipitation with helicase assay","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — helicase activity measured from chromatin-immunoprecipitated complex, CDK dependency shown, single lab","pmids":["12581157"],"is_preprint":false},{"year":2012,"finding":"Mcm10 plays a role in CMG helicase function independent of CMG assembly. In budding yeast, auxin-induced degradation of Mcm10 allows stable CMG assembly at origins, but subsequent CMG translocation, RPA loading, and intra-S checkpoint activation are severely diminished. Mcm10 chromatin association depends on S-CDK and CDC45.","method":"Auxin-inducible degron, chromatin immunoprecipitation, budding yeast","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — inducible protein degradation with epistasis ordering, single lab","pmids":["22032285"],"is_preprint":false},{"year":2007,"finding":"CDC45 protein is ubiquitylated and degraded via the proteasome pathway during terminal differentiation of human cells. Proteasome inhibitors decelerate CDC45 loss during differentiation. Multiple putative destruction boxes and a KEN-box suggest CDC45 is an APC/C substrate. CDC45 is not cleaved during apoptosis.","method":"Proteasome inhibitor treatment, immunoblotting (human cells)","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — proteasome inhibitor rescue only, no direct ubiquitylation assay, single lab","pmids":["17767920"],"is_preprint":false},{"year":2013,"finding":"Overexpression of CDC45 in Xenopus recapitulates c-Myc-induced replication phenotypes: increased density of early-replicating origins, elevated replication fork stalling/collapse, and DNA damage. CDC45 and GINS function downstream of Myc in regulating replication initiation.","method":"Xenopus egg extract, DNA fiber analysis, immunofluorescence, epistasis via overexpression","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression epistasis in cell-free system plus fiber analysis, single lab","pmids":["23643534"],"is_preprint":false},{"year":2023,"finding":"DONSON is required for CDC45 and GINS association with MCM2-7 (CMG assembly) during replication initiation in Xenopus egg extracts. DONSON interacts with the initiation factor TopBP1 in a CDK-dependent manner. DONSON also associates with the replisome during elongation.","method":"Xenopus egg extracts, immunodepletion, chromatin fractionation, co-immunoprecipitation","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunodepletion in cell-free system with epistasis, single lab","pmids":["37638758"],"is_preprint":false},{"year":2016,"finding":"In human cells, CDC45 overexpression fires at least twice as many origins but causes ~2-fold reduced fork elongation rate, pronounced fork asymmetry, S-phase arrest, accumulation of long ssDNA stretches (replication catastrophe), and ATM/Chk2-mediated H2AX phosphorylation, consistent with CDC45 being rate-limiting for origin firing.","method":"DNA fiber assay, flow cytometry, immunofluorescence, overexpression in human cell lines","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — DNA fiber analysis plus multiple cellular readouts, single lab","pmids":["26919204"],"is_preprint":false},{"year":2019,"finding":"Myc induces chromatin decondensation at targeted sites and directly promotes CDC45/GINS recruitment to resident MCMs, activating CMG helicases. Myc-Box II (MBII) and its interactors GCN5, Tip60, and TRRAP are required for chromatin unfolding and CDC45 recruitment. Myc and CDC45 physically interact.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, lac-repressor chromatin targeting, siRNA depletion (mammalian cells)","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction plus ChIP plus chromatin targeting assay, single lab","pmids":["30911685"],"is_preprint":false},{"year":2019,"finding":"DNAJA1 (Hsp40 family) stabilizes CDC45 protein and promotes cell cycle progression. KNK437 reduces DNAJA1 levels, leading to reduced CDC45 stability. E2F1 transcriptionally activates DNAJA1, which then stabilizes CDC45 to promote the cell cycle.","method":"siRNA knockdown, Western blotting, co-immunoprecipitation (human colorectal cancer cells)","journal":"Oncogene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP with limited mechanistic follow-up on direct stabilization mechanism","pmids":["31477839"],"is_preprint":false},{"year":2019,"finding":"CDC45 contains an initiation-specific function: temperature-sensitive CDC45 mutants (in the RecJ-like domain and IDR) are defective for CMG formation and replication initiation but not elongation. The IDR of CDC45 is required for its function when carrying lethal point mutations but CDC45 lacking the IDR entirely retains full function, indicating the IDR context matters for initiation.","method":"Site-directed mutagenesis, temperature-sensitive yeast genetics, CMG formation assay, in vivo replication assay (budding yeast)","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and biochemical characterization of multiple alleles, CMG formation assay, single lab","pmids":["30913274"],"is_preprint":false},{"year":2015,"finding":"Dpb11 (human TopBP1 ortholog) binds Mcm2-7 and competes with GINS for Mcm2-7 binding. Dpb11 can recruit CDC45 to Mcm2-7. ssDNA inhibits Dpb11-Mcm2-7 interaction, allowing GINS to displace Dpb11 and bind Mcm2-7 for CMG assembly.","method":"Purified protein binding assays, competition experiments, yeast genetics, ChIP (budding yeast)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — reconstituted with purified proteins plus in vivo genetic validation, single lab","pmids":["25659432"],"is_preprint":false},{"year":2013,"finding":"Human CDC45 directly interacts with all MCM2-7 subunits and with PSF2, PSF3, and SLD5 (GINS subunits), as well as RPA2, AND-1, and TopBP1 by immunoprecipitation. A considerable portion of CDC45 in nuclei is associated with nuclear scaffold structures (nuclease-resistant fraction) rather than at replication forks.","method":"Immunoprecipitation, chromatin fractionation, nuclease treatment, synchronized HeLa cells","journal":"Journal of biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP without direct reciprocal confirmation, single lab, localization without clear functional consequence","pmids":["23364835"],"is_preprint":false},{"year":2019,"finding":"PP2A exists in complex with CDC45 during DNA replication, and increased PP2A activity causes dissociation of CDC45 and polymerase alpha from the replisome, interrupting ongoing DNA replication and causing replication fork collapse.","method":"Co-immunoprecipitation, PP2A activator (small molecule), PP2A genetic loss-of-function, DNA replication assays (human cells)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus gain- and loss-of-function PP2A approaches, single lab","pmids":["31562245"],"is_preprint":false},{"year":2016,"finding":"Biallelic partial loss-of-function mutations in human CDC45 cause Meier-Gorlin syndrome and craniosynostosis. Mutations reduce full-length CDC45 transcript and protein levels in patient cells, consistent with reduced DNA replication rate and cell proliferation. CDC45 is thus functionally distinct from pre-RC MGS genes, implicating the pre-IC in MGS etiology.","method":"Whole-exome sequencing, RT-PCR splicing analysis, patient cell lines (protein quantification)","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient mutation identification with functional validation in patient cells, multiple families, single study","pmids":["27374770"],"is_preprint":false},{"year":2001,"finding":"CDC45 null mouse embryos fail to develop past implantation; inner cell mass shows impaired proliferation, establishing CDC45 as essential for mammalian post-implantation development and cell proliferation in vivo. Heterozygous mice develop normally, suggesting hemizygosity of CDC45 alone is insufficient to cause cardiac/craniofacial defects in DiGeorge syndrome.","method":"Gene targeting (knockout mouse), embryo culture, immunostaining","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — homozygous KO mouse with defined developmental phenotype, single lab","pmids":["11416137"],"is_preprint":false}],"current_model":"CDC45 is an essential eukaryotic DNA replication factor that, together with MCM2-7 and GINS, forms the CMG (Cdc45/Mcm2-7/GINS) replicative helicase complex: CDC45 and GINS bridge the MCM2/MCM5 gate to allosterically activate MCM2-7 ATPase and helicase activity, with CDC45 itself guarding the leading-strand template via its RecJ-like fold; CDC45 is loaded at origins in a DDK- and CDK-dependent, stepwise manner (requiring Sld3, Mcm10, TopBP1/Xmus101, and other factors), is rate-limiting for origin firing, travels with the replisome throughout elongation, loads RPA onto nascent ssDNA, bridges replicative polymerases to the helicase, and is targeted by checkpoint kinases (Chk1 displaces it from chromatin; Chk2 directly phosphorylates CMG subunits to inhibit helicase activity), while loss-of-function mutations cause Meier-Gorlin syndrome and craniosynostosis in humans."},"narrative":{"mechanistic_narrative":"CDC45 is an essential eukaryotic DNA replication factor that, together with MCM2-7 and GINS, constitutes the CMG (Cdc45/Mcm2-7/GINS) replicative helicase [PMID:16798881, PMID:20122406]. CDC45 and GINS bridge the Mcm2/Mcm5 gate of the helicase ring to form a topologically closed assembly, and this association elevates MCM2-7 ATP hydrolysis ~100-fold and converts the ring into a processive 3'-to-5' helicase that translocates on the leading-strand template [PMID:20122406, PMID:21378962, PMID:22474384]. CDC45 retains a RecJ-like (DHH-family) fold whose ssDNA-binding groove guards the leading strand within the CMG, and mutations in this fold diminish helicase activity [PMID:27189187, PMID:25561522, PMID:22147708]. CDC45 is loaded at origins in a stepwise, DDK- and CDK-dependent manner downstream of pre-RC assembly, requiring Mcm10, Sld3, TopBP1/Dpb11, and DONSON, with GINS displacing Sld3/Dpb11 to mature the CMS intermediate into the active CMG [PMID:11296242, PMID:11864598, PMID:16990792, PMID:21362622, PMID:37638758, PMID:25659432]; the timing of CDC45 origin association is the key determinant of origin firing time and is rate-limiting for initiation [PMID:22169533, PMID:26919204]. Beyond unwinding, CDC45 coordinates polymerase recruitment, tethering DNA polymerase alpha onto MCM via direct binding, supporting sequential loading of RPA, pol alpha, and PCNA, and catalytically loading RPA onto nascent ssDNA [PMID:9755170, PMID:10886370, PMID:28100698, PMID:10518787]. CDC45 travels with the replisome throughout elongation and acts as a checkpoint hub: Chk2 phosphorylates CMG subunits to inhibit helicase activity, Chk1 displaces CDC45 from chromatin, and the CDC45 disordered loop recruits Rad53/checkpoint kinase to stalled forks [PMID:15329670, PMID:22853956, PMID:16912045, PMID:30595439]. Biallelic partial loss-of-function mutations in CDC45 cause Meier-Gorlin syndrome and craniosynostosis, and CDC45 is essential for mammalian post-implantation development [PMID:27374770, PMID:11416137].","teleology":[{"year":1997,"claim":"Established CDC45 as a genetically required initiation factor acting after START and downstream of pre-RC components, answering where in the cell cycle CDC45 functions.","evidence":"Yeast genetics, synthetic lethality with ORC/MCM alleles, 2D gel origin firing analysis, and CDC7/Dbf4 epistasis","pmids":["9001208","9356482"],"confidence":"High","gaps":["Did not define a biochemical activity for CDC45","Mechanism of CDC45-dependent origin activation unresolved"]},{"year":2000,"claim":"Demonstrated that CDC45 drives the origin-unwinding step and orders the loading of downstream replication factors, distinguishing helicase activation from polymerase recruitment.","evidence":"Xenopus egg extract immunodepletion, supercoiling/unwinding assays, chromatin fractionation, and aphidicolin uncoupling","pmids":["10882098","10886370","9755170"],"confidence":"High","gaps":["Did not identify the helicase enzyme itself","How CDC45 physically couples to MCM unclear at this stage"]},{"year":2006,"claim":"Identified CDC45-MCM2-7-GINS as a single stable complex (CMG) that is the eukaryotic replicative helicase, resolving the long-standing question of the replicative helicase identity.","evidence":"Immunoaffinity purification with MS, in vitro helicase assay on purified Drosophila CMG, and RNAi knockdown","pmids":["16531994","16798881","16483939"],"confidence":"High","gaps":["Did not define how GINS/CDC45 mechanistically activate MCM","Strand handling within the helicase unknown"]},{"year":2010,"claim":"Showed mechanistically that CDC45 and GINS association activates the MCM ATPase and helicase, quantifying the activation and demonstrating cooperative complex formation.","evidence":"Reconstitution from recombinant Drosophila proteins with ATPase, helicase, and pulldown assays","pmids":["20122406"],"confidence":"High","gaps":["Structural basis of activation not yet visualized","Did not address leading vs lagging strand routing"]},{"year":2011,"claim":"Provided the structural explanation for CMG activation, showing GINS and CDC45 bridge the Mcm2/Mcm5 gate to close the helicase ring.","evidence":"Single-particle EM of MCM2-7 and CMG with functional interpretation","pmids":["21378962"],"confidence":"High","gaps":["Resolution insufficient for atomic detail of CDC45","DNA path within channel not directly resolved"]},{"year":2013,"claim":"Defined CDC45 as an ssDNA-binding RecJ/DHH-fold protein that engages 3'-protruding and forked structures, linking its architecture to a strand-handling role.","evidence":"Recombinant human/yeast CDC45 with EMSA, AFM, SPR, SAXS binding assays and in vivo mutant phenotypes","pmids":["22147708","24293646","23382391"],"confidence":"Medium","gaps":["Most DHH catalytic residues not conserved; no nuclease activity demonstrated","Single-lab biochemistry"]},{"year":2015,"claim":"Established that CDC45 guards the leading strand within the CMG, defining its role in strand exclusion during unwinding.","evidence":"DNA-protein crosslinking, site-directed mutagenesis, and helicase assays on Drosophila CMG","pmids":["25561522"],"confidence":"High","gaps":["Physiological consequence of leading-strand guarding in vivo not fully established"]},{"year":2016,"claim":"Resolved the atomic structure of human CDC45 and connected disease-relevant residues to CMG assembly mechanism.","evidence":"X-ray crystallography at 2.1 A with structure-guided mutagenesis and EM integration","pmids":["27189187"],"confidence":"High","gaps":["Conformational changes upon CMG incorporation not captured","Function of the helical insertion only inferred"]},{"year":2021,"claim":"Defined how DDK phosphorylation of MCM tails templates a two-stage CMG assembly via Cdc45-tail-GINS intermediates, answering how CDC45 recruitment is regulated kinetically.","evidence":"Single-molecule fluorescence reconstitution with DDK phosphorylation (building on Sld3/GINS competition and ordered-loading studies)","pmids":["33616038","21362622","25659432","16990792"],"confidence":"High","gaps":["The inefficient second step's molecular determinants remain undefined","In vivo correspondence of CtG intermediates not directly shown"]},{"year":2017,"claim":"Showed CDC45 catalytically loads RPA onto nascent ssDNA, extending its role beyond unwinding to coordinating ssDNA protection at the fork.","evidence":"Pull-down, SPR, and real-time RPA-ssDNA binding assays with human recombinant proteins","pmids":["28100698","10518787","17573775"],"confidence":"Medium","gaps":["Catalytic mechanism of RPA handoff not structurally resolved","Single-lab kinetic study"]},{"year":2018,"claim":"Identified CDC45 as a checkpoint scaffold recruiting Rad53/checkpoint kinase and linked a CDC45 disordered loop to fork stabilization and a disease mutation.","evidence":"Co-IP, in vitro phosphorylation, genetic epistasis, and Meier-Gorlin mutation analysis (with Chk1/Chk2 displacement studies)","pmids":["30595439","22853956","16912045"],"confidence":"Medium","gaps":["How checkpoint engagement at CDC45 is timed relative to elongation unclear","Single-lab Co-IP for several interactions"]},{"year":2016,"claim":"Linked CDC45 dosage to genome stability and established disease causation, showing CDC45 is rate-limiting for origin firing and causative for Meier-Gorlin syndrome.","evidence":"DNA fiber and cellular assays under CDC45 overexpression, plus whole-exome sequencing and patient-cell functional validation, with KO mouse lethality","pmids":["26919204","27374770","53"],"confidence":"Medium","gaps":["Tissue-specific basis of MGS/craniosynostosis phenotype unexplained","Quantitative link between CDC45 level and origin number incompletely defined"]},{"year":null,"claim":"How CDC45 abundance, post-translational regulation (PP2A, ubiquitin/APC-C, DNAJA1 chaperone, Myc-driven recruitment) and checkpoint signaling are integrated to set origin firing rates and protect forks across differentiated tissues remains unresolved.","evidence":"Not established in the available corpus","pmids":[],"confidence":"Low","gaps":["No unified model of CDC45 dosage control in vivo","Direct ubiquitylation and APC/C targeting not biochemically demonstrated","Tissue specificity of regulation unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[13,14,15]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[1,2,11,12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8,9,16,33]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,34,50]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[6,27,40]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[32,50]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[1,2,11,19]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[18,24,53]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[17,27,28]}],"complexes":["CMG helicase (Cdc45/Mcm2-7/GINS)","Replisome progression complex (RPC)","Cdc45-MCM2-7-Sld3 (CMS) intermediate"],"partners":["MCM7","GINS (PSF2)","POLA1","RPA","SLD3","TOPBP1","MCM10","DONSON"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75419","full_name":"Cell division control protein 45 homolog","aliases":["PORC-PI-1"],"length_aa":566,"mass_kda":65.6,"function":"Required for initiation of chromosomal DNA replication. Core component of CDC45-MCM-GINS (CMG) helicase, the molecular machine that unwinds template DNA during replication, and around which the replisome is built","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/O75419/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CDC45","classification":"Common Essential","n_dependent_lines":1207,"n_total_lines":1208,"dependency_fraction":0.9991721854304636},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CDC45","total_profiled":1310},"omim":[{"mim_id":"617063","title":"MEIER-GORLIN SYNDROME 7; MGORS7","url":"https://www.omim.org/entry/617063"},{"mim_id":"614448","title":"GEMININ COILED-COIL DOMAIN-CONTAINING PROTEIN; GMNC","url":"https://www.omim.org/entry/614448"},{"mim_id":"613298","title":"TOPBP1-INTERACTING CHECKPOINT AND REPLICATION REGULATOR; TICRR","url":"https://www.omim.org/entry/613298"},{"mim_id":"611428","title":"DOWNSTREAM NEIGHBOR OF SON; DONSON","url":"https://www.omim.org/entry/611428"},{"mim_id":"610909","title":"MINICHROMOSOME MAINTENANCE COMPLEX-BINDING PROTEIN; MCMBP","url":"https://www.omim.org/entry/610909"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Centrosome","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":24.0},{"tissue":"lymphoid tissue","ntpm":19.0},{"tissue":"testis","ntpm":26.6}],"url":"https://www.proteinatlas.org/search/CDC45"},"hgnc":{"alias_symbol":[],"prev_symbol":["CDC45L2","CDC45L"]},"alphafold":{"accession":"O75419","domains":[{"cath_id":"-","chopping":"215-426","consensus_level":"medium","plddt":94.6683,"start":215,"end":426},{"cath_id":"3.10.310","chopping":"438-566","consensus_level":"high","plddt":94.4552,"start":438,"end":566}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75419","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75419-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75419-F1-predicted_aligned_error_v6.png","plddt_mean":92.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CDC45","jax_strain_url":"https://www.jax.org/strain/search?query=CDC45"},"sequence":{"accession":"O75419","fasta_url":"https://rest.uniprot.org/uniprotkb/O75419.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75419/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75419"}},"corpus_meta":[{"pmid":"16531994","id":"PMC_16531994","title":"GINS 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GINS is essential for maintaining the association of CDC45 with MCM within RPCs after initiation, and RPCs also contain Mrc1, Tof1-Csm3, FACT, Ctf4, Mcm10, and topoisomerase I.\",\n      \"method\": \"Immunoaffinity purification, mass spectrometry, chromatin immunoprecipitation (budding yeast)\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-purification with MS identification, replicated by multiple labs in same year\",\n      \"pmids\": [\"16531994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CDC45, MCM2-7, and GINS form the CMG (Cdc45/Mcm2-7/GINS) complex, which is the eukaryotic replicative DNA helicase. The purified complex from Drosophila embryo extracts has ATP-dependent DNA helicase activity. RNAi knockdown of GINS or CDC45 blocks S-phase transition.\",\n      \"method\": \"Immunoaffinity purification, in vitro helicase assay, RNAi knockdown (Drosophila)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro helicase assay with purified complex, corroborated by RNAi functional assay, replicated by multiple labs\",\n      \"pmids\": [\"16798881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Association of CDC45 and GINS with MCM2-7 activates the helicase: ATP hydrolysis rates are elevated ~100-fold, helicase activity is robust on circular templates, and DNA affinity is improved. GINS binds specifically to MCM4. All pairwise associations among GINS, MCMs, and CDC45 are detectable but tight association requires the full CMG.\",\n      \"method\": \"Reconstitution with recombinant Drosophila proteins, ATPase assay, in vitro helicase assay, pulldown\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted CMG from recombinant components, multiple orthogonal biochemical assays, single rigorous study\",\n      \"pmids\": [\"20122406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Cryo-EM structures of MCM2-7 and the CMG complex reveal that GINS and CDC45 bridge the Mcm2/Mcm5 gap in the helicase ring, forming a topologically closed assembly. Nucleotide binding further seals the ring, partitioning the central channel into two pores. This explains how GINS and CDC45 activate Mcm2-7 helicase.\",\n      \"method\": \"Single-particle electron microscopy, structural analysis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure of CMG with functional interpretation, landmark structural study\",\n      \"pmids\": [\"21378962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of human CDC45 at 2.1 Å confirms evolutionary relationship to bacterial RecJ nuclease (DHH family). Key features include: long-range N-C terminal DHH domain interaction blocking the DNA-binding groove, and a helical insertion poised for replisome interactions. Mutational analysis validated the mechanism of CDC45 association with the MCM ring and GINS co-activator critical for CMG assembly.\",\n      \"method\": \"X-ray crystallography (2.1 Å), structure-guided mutagenesis, EM data integration\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic resolution crystal structure with mutagenesis validation, multiple orthogonal methods in single study\",\n      \"pmids\": [\"27189187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CDC45 is required for origin unwinding during replication initiation in Xenopus. CDC45 binds chromatin upstream of RPA and DNA polymerase alpha. When CDC45 is present but DNA pol alpha is inhibited, helicase activity becomes uncoupled, demonstrating that CDC45 drives the unwinding step.\",\n      \"method\": \"Xenopus egg extract replication assay, immunodepletion, supercoiling assay, chromatin fractionation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunodepletion in cell-free system with multiple functional readouts, independently replicated\",\n      \"pmids\": [\"10882098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"At paused replication forks in Xenopus, MCM2-7, CDC45, and GINS are enriched at the unwinding site even when polymerase is inhibited by aphidicolin, establishing these three as core components of the 'unwindosome' that separates DNA strands at the replication fork.\",\n      \"method\": \"Biotin-streptavidin fork-pausing assay, chromatin immunoprecipitation, Xenopus egg extracts\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct chromatin localization at defined fork position, multiple replication factors tested, Xenopus cell-free system\",\n      \"pmids\": [\"16483939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MCM7 and CDC45 are required throughout replication elongation (not just initiation) in vertebrates. Antibody neutralization of CDC45 or MCM7 after significant DNA synthesis had already occurred blocked further synthesis and abolished helicase-dependent chromosome unwinding (uncoupled by aphidicolin), establishing CDC45 as a helicase co-factor essential for elongation.\",\n      \"method\": \"Xenopus egg extract, antibody neutralization, aphidicolin uncoupling assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — antibody depletion at defined cell cycle stages, multiple functional readouts, Xenopus system\",\n      \"pmids\": [\"15329670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Xenopus CDC45 loads DNA polymerase alpha onto chromatin at replication initiation. CDC45 physically interacts with DNA polymerase alpha in egg extracts, associates with chromatin only after nuclear formation in an S-phase CDK-dependent manner, and co-localizes with pol alpha in S-phase nuclei.\",\n      \"method\": \"Xenopus egg extract, co-immunoprecipitation, chromatin fractionation, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP plus functional depletion in cell-free system, multiple orthogonal methods\",\n      \"pmids\": [\"9755170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CDC45 is essential for the sequential chromatin loading of RPA, DNA pol alpha, and PCNA at replication initiation in Xenopus. CDC45 forms a stable complex with either MCM or DNA pol alpha on chromatin. DNA pol epsilon loading requires CDC45 but not pol alpha, suggesting a dual role in DNA unwinding and polymerase recruitment.\",\n      \"method\": \"Xenopus egg extract, immunodepletion, chromatin fractionation, co-immunoprecipitation\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunodepletion with multiple downstream readouts in cell-free system\",\n      \"pmids\": [\"10886370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CDC45 forms a complex with Sld3 throughout the cell cycle in S. cerevisiae. Their origin associations are mutually dependent. In sld3 mutants, the Sld3-CDC45 interaction and the CDC45-MCM2 interaction are both reduced. RPA does not associate with origins in the absence of Sld3, showing that the Sld3-CDC45 complex is prerequisite for origin unwinding.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, two-hybrid, genetic analysis (budding yeast)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus ChIP plus genetic epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"11296242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The human CMG complex (CDC45/MCM2-7/GINS) purified from baculovirus-infected Sf9 cells has DNA helicase activity that: requires forked DNA structures for maximal activity; translocates 3' to 5' on the leading strand template; unwinds up to 500 bp; and, together with DNA pol epsilon, supports leading-strand synthesis >10 kb.\",\n      \"method\": \"Baculovirus expression, in vitro helicase assay, ATPase assay, rolling circle DNA synthesis assay (human CMG)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted human CMG, multiple in vitro biochemical assays establishing directionality and leading-strand synthesis\",\n      \"pmids\": [\"22474384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CDC45 guards the leading strand within the CMG: cross-linking studies show the leading strand contacts CDC45 when the MCM2/5 gate is open, but the lagging strand does not pass through the side channel. Mutations in the RecJ-like fold of CDC45 that ablate this leading-strand interaction diminish helicase activity.\",\n      \"method\": \"DNA-protein crosslinking, site-directed mutagenesis, in vitro helicase assay (Drosophila CMG)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural crosslinking with functional mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"25561522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CDC45 binds single-stranded DNA (ssDNA) with a structure similar to RecJ, demonstrating evolutionary relationship to DHH phosphoesterase family. Biochemical and SAXS data confirm only a subset of the Mn2+-coordinating residues are conserved, but the protein retains ssDNA (not dsDNA) binding activity.\",\n      \"method\": \"Recombinant human CDC45, SAXS, ssDNA binding assays, bioinformatics\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical assay plus structural SAXS, single lab, consistent with evolutionary prediction\",\n      \"pmids\": [\"22147708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human CDC45 binds long ssDNA (≥40 nt) and preferentially binds 3'-protruding strands, Y-shaped DNA, bubbles, and D-loops with higher affinity than short oligonucleotides. CDC45 slides on DNA with 3'-5' polarity, suggesting it acts as a molecular wedge to initiate strand displacement.\",\n      \"method\": \"Recombinant human CDC45, EMSA, AFM, SPR, SRCD, SAXS\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal in vitro binding assays, single lab\",\n      \"pmids\": [\"24293646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CDC45 from budding yeast binds tightly to long (≥40 nt) ssDNA; 60-mer ssDNA disrupts the CDC45-MCM2-7 interaction. A CDC45 mutant unable to bind ssDNA causes helicase uncoupling from the polymerase under replication stress (hydroxyurea), with excess RPA accumulating near origins, demonstrating that CDC45-ssDNA interaction is required to stall the helicase during replication stress.\",\n      \"method\": \"Purified protein binding assays, site-directed mutagenesis, yeast genetics, chromatin immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding plus in vivo mutant phenotype, single lab\",\n      \"pmids\": [\"23382391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human CDC45 actively loads RPA onto nascent ssDNA in a catalytic manner. CDC45 forms a complex with RPA and stabilizes the 8-10 nt RPA binding mode; interaction requires the RPA70A subdomain. RPA dissociates when it covers a 30-mer. CDC45 facilitates ordered RPA deposition on ssDNA at the replication fork.\",\n      \"method\": \"Pull-down assay, surface plasmon resonance, real-time RPA-ssDNA binding assay (human recombinant proteins)\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with kinetic analysis, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"28100698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Checkpoint kinase Chk2 (but not Chk1) directly inhibits CMG helicase activity in vitro by phosphorylating MCM3, MCM4, and GINS subunit Psf2. Phosphatase treatment of CMG stimulates helicase activity. Ionizing radiation in Drosophila embryos causes hyperphosphorylation of Psf2 within the active helicase complex in vivo.\",\n      \"method\": \"In vitro kinase assay, helicase assay with recombinant Drosophila CMG, mass spectrometry, Drosophila embryo irradiation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with kinase, phosphorylation site mapping by MS, in vivo confirmation in embryos\",\n      \"pmids\": [\"22853956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CDC45 functions in late G1 phase after START and prior to DNA synthesis to trigger initiation at replication origins. CDC45 and CDC7/Dbf4 kinase are mutually dependent for function; cells defective in CDC45 cannot activate prereplicative complexes.\",\n      \"method\": \"Yeast genetics, cell cycle synchronization, epistasis analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis, cell cycle timing experiments, single lab\",\n      \"pmids\": [\"9356482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CDC45 is essential for DNA replication initiation in S. cerevisiae. It genetically interacts with MCM genes (CDC46, CDC47, CDC54) and is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. Origins fire less frequently in cdc45-1 cells, establishing CDC45 as functioning with ORC and MCM proteins in replication initiation.\",\n      \"method\": \"Yeast genetics, complementation, 2D gel origin firing analysis, synthetic lethality\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic interaction tests, direct 2D gel analysis of origin firing\",\n      \"pmids\": [\"9001208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cdc7-Dbf4 kinase (DDK)-dependent phosphorylation of MCM4 N-terminal residues stimulates CDC45 association with chromatin. Deletion of MCM4 N-terminal 150 aa causes growth inhibition, and combined alanine substitution/deletion of N-terminal segments of MCM2, MCM4, and MCM6 leads to non-viable phenotype, indicating redundant but essential roles for these DDK-target sites in CDC45 loading.\",\n      \"method\": \"Chromatin fractionation, phospho-specific antibodies, SDS-PAGE mobility shift, Cdc7 conditional KO mouse ES cells, siRNA, mutagenesis (mammalian cells)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-specific antibodies, genetic rescue, conditional KO confirmation, single lab\",\n      \"pmids\": [\"17046832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Xenopus MCM10 binds chromatin after MCM2-7 but upstream of CDC45. In the absence of MCM10, CDC45 binding, RPA binding, and origin unwinding (supercoiling) are all blocked, placing MCM10 as an essential intermediate between pre-RC assembly and CDC45 loading.\",\n      \"method\": \"Xenopus egg extract, immunodepletion, chromatin fractionation, supercoiling assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunodepletion in cell-free system with multiple downstream readouts, epistasis ordering\",\n      \"pmids\": [\"11864598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Protein phosphatase 2A (PP2A) is required for CDC45 loading onto the pre-RC in Xenopus. PP2A depletion or okadaic acid treatment abolishes CDC45 loading, origin unwinding, and downstream RPA and pol alpha loading. PP2A acts on a soluble factor (not CDC45 itself or pre-RC components) to enable CDC45 loading.\",\n      \"method\": \"Xenopus egg extract, PP2A immunodepletion, okadaic acid inhibition, chromatin fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunodepletion plus chemical inhibition, epistasis ordering, single lab\",\n      \"pmids\": [\"12185086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Xmus101 (TOPBP1 ortholog) is required for loading CDC45 onto origins in Xenopus. Xmus101 chromatin association depends on ORC but is independent of MCM2-7 and S-CDK, defining a parallel ORC-dependent pathway for CDC45 loading distinct from the MCM2-7 pathway.\",\n      \"method\": \"Xenopus egg extract, immunodepletion, chromatin fractionation, epistasis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunodepletion with clear epistasis ordering, single lab\",\n      \"pmids\": [\"12438414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Origin association timing of CDC45 (together with Sld3 and Sld7) is the key determinant of origin firing time in budding yeast. CDC45 associates with early-firing origins in G1 in a DDK-dependent manner; increased dosage of Sld3/Sld7/CDC45 allows late origins to fire earlier, as does increased DDK dosage.\",\n      \"method\": \"Chromatin immunoprecipitation, dosage analysis, genetic overexpression (budding yeast)\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus overexpression genetics, single lab, clear functional readout\",\n      \"pmids\": [\"22169533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In fission yeast, Sld3 is loaded at origins upstream of GINS, Cut5, and CDC45. DDK but not CDK is required for Sld3 loading, while CDC45 loading requires both kinases. GINS integrity is required for CDC45 loading but not Sld3 loading, establishing the ordered assembly: Sld3 → GINS/Cut5 → CDC45.\",\n      \"method\": \"Chromatin immunoprecipitation, temperature-sensitive mutant analysis, pull-down (fission yeast)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP epistasis ordering with multiple mutants, single lab\",\n      \"pmids\": [\"16990792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Assembly of the human CMG complex (CDC45-MCM2-7-GINS) in HeLa cells requires CDK activity, CDC7 kinase, and the additional proteins RecQL4, Ctf4/And-1, and Mcm10. CMG interactions are only observed after G1/S transition. Depletion of TopBP1 did not significantly affect CMG complex formation in human cells.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC) in HeLa cells, siRNA depletion, CDK inhibitor treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BiFC in human cells plus siRNA epistasis, single lab\",\n      \"pmids\": [\"19805216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Chk1-mediated S-phase checkpoint targets CDC45 via a Cdc25A/CDK2-independent mechanism. BPDE-induced DNA damage causes Chk1-dependent reduction of chromatin-associated CDC45 (not soluble CDC45) and disrupts the CDC45-MCM7 interaction at the beta-globin replication origin, without affecting MCM7, MCM10, or PCNA chromatin binding.\",\n      \"method\": \"Chromatin fractionation, co-immunoprecipitation, chromatin immunoprecipitation, Chk1 inhibitor UCN-01 (human cells)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP at specific origin plus co-IP plus specific inhibitor rescue, single lab\",\n      \"pmids\": [\"16912045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CDC45 targets checkpoint kinase Rad53 to replication complexes via FHA-domain interaction with phosphorylated motifs in an intrinsically disordered loop of Cdc45. This interaction is necessary for Rad53-mediated inhibition of origin firing through Sld3, and also for stabilizing stalled forks. A CDC45 mutation found in Meier-Gorlin syndrome disrupts the Rad53 interaction.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphorylation, genetic epistasis, budding yeast\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus genetic epistasis plus disease mutation validation, single lab\",\n      \"pmids\": [\"30595439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GINS and Sld3 compete with each other for binding to both MCM2-7 and CDC45. Purified proteins form either a Cdc45-MCM2-7-Sld3 (CMS) or Cdc45-MCM2-7-GINS (CMG) complex with 1:1:1 stoichiometry. The data suggest GINS displaces Sld3 at the origin to activate the replication fork helicase.\",\n      \"method\": \"Purified recombinant protein binding assays, size exclusion chromatography, competition assays (budding yeast)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted complexes from purified components, stoichiometry determination, single lab\",\n      \"pmids\": [\"21362622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DDK regulates CMG formation via a two-stage mechanism: DDK phosphorylation of MCM2-7 N-terminal tails recruits Cdc45 and GINS to form Cdc45-tail-GINS (CtG) intermediates. Higher DDK phosphorylation increases CtG multiplicity per MCM2-7, and higher CtG numbers increase the frequency of CMG formation in a second, inefficient step.\",\n      \"method\": \"Single-molecule fluorescence microscopy, in vitro reconstitution, DDK phosphorylation assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — single-molecule resolution biochemical reconstitution, mechanistic ordering of two-stage assembly, single rigorous study\",\n      \"pmids\": [\"33616038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human CDC45 interacts with Claspin, RPA, and DNA polymerase delta maximally during S phase. UV-induced DNA damage reduces CDC45-Claspin complex formation without affecting CDC45-RPA interaction, and this dissociation occurs upstream of ATR activation in the S-phase checkpoint.\",\n      \"method\": \"Co-immunoprecipitation, synchronized HeLa cells, UV treatment, kinase inhibitors\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP approach, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"23910567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human CDC45 co-localizes with active replication sites during S phase and interacts with DNA polymerase delta, DNA polymerase epsilon, GINS subunit Psf2, and MCM5/7 subunits, suggesting CDC45 bridges replicative polymerases with the MCM helicase in the elongation complex.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization (human cells)\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus co-localization, single lab, no functional manipulation\",\n      \"pmids\": [\"17573775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human CDC45 directly binds hMCM7 and the p70 subunit of DNA polymerase alpha in vitro, supporting a role as molecular tether for loading pol alpha onto the replication complex via MCM7.\",\n      \"method\": \"In vitro binding assay (direct interaction), pull-down (human proteins)\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro binding with recombinant proteins, single lab, two interaction partners characterized\",\n      \"pmids\": [\"10518787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human CDC45 (CDC45L) co-immunoprecipitates with human ORC2 from cell extracts; the protein associates with the nuclear fraction in G1 but this association becomes labile as S phase progresses, consistent with a role in replication initiation.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation (human cells)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP, single lab, no functional manipulation\",\n      \"pmids\": [\"9660782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Targeting CDC45 to specific chromosomal sites in mammalian cells induces large-scale chromatin decondensation correlated with histone H1 phosphorylation. CDC45 recruits CDK2 to these sites; CDK2 activity is required for decondensation. CDC45, CDK2, cyclin A, and phospho-H1 physically interact and associate with chromatin during S phase.\",\n      \"method\": \"Lac-repressor chromatin targeting, immunofluorescence, CDK2 inhibitors, co-immunoprecipitation (mammalian cells)\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct chromatin targeting with functional readout, co-IP, inhibitor validation, single lab\",\n      \"pmids\": [\"15753125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Human TopBP1 directly interacts with CDC45 in vitro and in vivo, with this interaction occurring exclusively at the G1/S boundary. The first and second BRCT domains of TopBP1 mediate binding to CDC45, and overexpression of the sixth BRCT domain reduces CDC45 chromatin loading.\",\n      \"method\": \"GST pull-down, co-immunoprecipitation, deletion mutant analysis, yeast/mammalian one-hybrid, chromatin fractionation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding (GST pull-down) plus co-IP plus functional chromatin loading readout, single lab\",\n      \"pmids\": [\"17887956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"DUE-B (DNA unwinding element binding protein) interacts with CDC45 and TopBP1 in cell extracts and baculovirus-expressed proteins. DUE-B and CDC45 co-localize at active replication origins. DUE-B immunodepletion in Xenopus egg extracts blocks replication and CDC45 (and a fraction of TopBP1) loading onto chromatin.\",\n      \"method\": \"Co-immunoprecipitation, baculovirus co-expression, chromatin immunoprecipitation, Xenopus immunodepletion\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus Xenopus depletion with functional readout, single lab\",\n      \"pmids\": [\"20065034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human Ctf4 (hCtf4) forms a complex with the CMG helicase in vitro (purified proteins), in Sf9 cells, and from HeLa chromatin. hCtf4 is a homodimer that acts as a platform linking pol alpha to CMG. The hCtf4-CMG complex retains helicase activity with greater salt resistance than CMG alone. Stability of hCtf4-CMG depends on interactions with multiple CMG components.\",\n      \"method\": \"In vitro binding with purified proteins, co-infection Sf9 cells, HeLa chromatin immunoprecipitation, helicase assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted from purified proteins plus in cellulo validation plus functional helicase assay, multiple orthogonal methods\",\n      \"pmids\": [\"24255107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Fission yeast Sna41 (CDC45 ortholog) facilitates loading of DNA pol alpha onto MCM proteins in vivo. Sna41 interacts with pol alpha throughout the cell cycle and with Mcm6 in chromatin fractions at G1-S. In a sna41 initiation-defective mutant, pol alpha does not interact with MCM6, establishing CDC45 as essential for pol alpha-MCM association.\",\n      \"method\": \"In vivo tagged protein co-immunoprecipitation, chromatin fractionation, fission yeast genetics\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo tagged proteins, chromatin fractionation, conditional mutant epistasis, single lab\",\n      \"pmids\": [\"11344166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Following replication initiation in Xenopus, CDC45 and all six MCM subunits form a tight complex on chromatin in a CDK-dependent manner. This MCM-CDC45 chromatin complex has DNA helicase activity, which requires both CDK activity and CDC45, providing direct evidence for CDC45 as a helicase co-factor in vivo.\",\n      \"method\": \"Xenopus egg extract, denaturing immunoprecipitation, chromatin immunoprecipitation with helicase assay\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — helicase activity measured from chromatin-immunoprecipitated complex, CDK dependency shown, single lab\",\n      \"pmids\": [\"12581157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Mcm10 plays a role in CMG helicase function independent of CMG assembly. In budding yeast, auxin-induced degradation of Mcm10 allows stable CMG assembly at origins, but subsequent CMG translocation, RPA loading, and intra-S checkpoint activation are severely diminished. Mcm10 chromatin association depends on S-CDK and CDC45.\",\n      \"method\": \"Auxin-inducible degron, chromatin immunoprecipitation, budding yeast\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — inducible protein degradation with epistasis ordering, single lab\",\n      \"pmids\": [\"22032285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CDC45 protein is ubiquitylated and degraded via the proteasome pathway during terminal differentiation of human cells. Proteasome inhibitors decelerate CDC45 loss during differentiation. Multiple putative destruction boxes and a KEN-box suggest CDC45 is an APC/C substrate. CDC45 is not cleaved during apoptosis.\",\n      \"method\": \"Proteasome inhibitor treatment, immunoblotting (human cells)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — proteasome inhibitor rescue only, no direct ubiquitylation assay, single lab\",\n      \"pmids\": [\"17767920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Overexpression of CDC45 in Xenopus recapitulates c-Myc-induced replication phenotypes: increased density of early-replicating origins, elevated replication fork stalling/collapse, and DNA damage. CDC45 and GINS function downstream of Myc in regulating replication initiation.\",\n      \"method\": \"Xenopus egg extract, DNA fiber analysis, immunofluorescence, epistasis via overexpression\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression epistasis in cell-free system plus fiber analysis, single lab\",\n      \"pmids\": [\"23643534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DONSON is required for CDC45 and GINS association with MCM2-7 (CMG assembly) during replication initiation in Xenopus egg extracts. DONSON interacts with the initiation factor TopBP1 in a CDK-dependent manner. DONSON also associates with the replisome during elongation.\",\n      \"method\": \"Xenopus egg extracts, immunodepletion, chromatin fractionation, co-immunoprecipitation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunodepletion in cell-free system with epistasis, single lab\",\n      \"pmids\": [\"37638758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In human cells, CDC45 overexpression fires at least twice as many origins but causes ~2-fold reduced fork elongation rate, pronounced fork asymmetry, S-phase arrest, accumulation of long ssDNA stretches (replication catastrophe), and ATM/Chk2-mediated H2AX phosphorylation, consistent with CDC45 being rate-limiting for origin firing.\",\n      \"method\": \"DNA fiber assay, flow cytometry, immunofluorescence, overexpression in human cell lines\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — DNA fiber analysis plus multiple cellular readouts, single lab\",\n      \"pmids\": [\"26919204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Myc induces chromatin decondensation at targeted sites and directly promotes CDC45/GINS recruitment to resident MCMs, activating CMG helicases. Myc-Box II (MBII) and its interactors GCN5, Tip60, and TRRAP are required for chromatin unfolding and CDC45 recruitment. Myc and CDC45 physically interact.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, lac-repressor chromatin targeting, siRNA depletion (mammalian cells)\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction plus ChIP plus chromatin targeting assay, single lab\",\n      \"pmids\": [\"30911685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DNAJA1 (Hsp40 family) stabilizes CDC45 protein and promotes cell cycle progression. KNK437 reduces DNAJA1 levels, leading to reduced CDC45 stability. E2F1 transcriptionally activates DNAJA1, which then stabilizes CDC45 to promote the cell cycle.\",\n      \"method\": \"siRNA knockdown, Western blotting, co-immunoprecipitation (human colorectal cancer cells)\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP with limited mechanistic follow-up on direct stabilization mechanism\",\n      \"pmids\": [\"31477839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CDC45 contains an initiation-specific function: temperature-sensitive CDC45 mutants (in the RecJ-like domain and IDR) are defective for CMG formation and replication initiation but not elongation. The IDR of CDC45 is required for its function when carrying lethal point mutations but CDC45 lacking the IDR entirely retains full function, indicating the IDR context matters for initiation.\",\n      \"method\": \"Site-directed mutagenesis, temperature-sensitive yeast genetics, CMG formation assay, in vivo replication assay (budding yeast)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and biochemical characterization of multiple alleles, CMG formation assay, single lab\",\n      \"pmids\": [\"30913274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Dpb11 (human TopBP1 ortholog) binds Mcm2-7 and competes with GINS for Mcm2-7 binding. Dpb11 can recruit CDC45 to Mcm2-7. ssDNA inhibits Dpb11-Mcm2-7 interaction, allowing GINS to displace Dpb11 and bind Mcm2-7 for CMG assembly.\",\n      \"method\": \"Purified protein binding assays, competition experiments, yeast genetics, ChIP (budding yeast)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted with purified proteins plus in vivo genetic validation, single lab\",\n      \"pmids\": [\"25659432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human CDC45 directly interacts with all MCM2-7 subunits and with PSF2, PSF3, and SLD5 (GINS subunits), as well as RPA2, AND-1, and TopBP1 by immunoprecipitation. A considerable portion of CDC45 in nuclei is associated with nuclear scaffold structures (nuclease-resistant fraction) rather than at replication forks.\",\n      \"method\": \"Immunoprecipitation, chromatin fractionation, nuclease treatment, synchronized HeLa cells\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP without direct reciprocal confirmation, single lab, localization without clear functional consequence\",\n      \"pmids\": [\"23364835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PP2A exists in complex with CDC45 during DNA replication, and increased PP2A activity causes dissociation of CDC45 and polymerase alpha from the replisome, interrupting ongoing DNA replication and causing replication fork collapse.\",\n      \"method\": \"Co-immunoprecipitation, PP2A activator (small molecule), PP2A genetic loss-of-function, DNA replication assays (human cells)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus gain- and loss-of-function PP2A approaches, single lab\",\n      \"pmids\": [\"31562245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Biallelic partial loss-of-function mutations in human CDC45 cause Meier-Gorlin syndrome and craniosynostosis. Mutations reduce full-length CDC45 transcript and protein levels in patient cells, consistent with reduced DNA replication rate and cell proliferation. CDC45 is thus functionally distinct from pre-RC MGS genes, implicating the pre-IC in MGS etiology.\",\n      \"method\": \"Whole-exome sequencing, RT-PCR splicing analysis, patient cell lines (protein quantification)\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient mutation identification with functional validation in patient cells, multiple families, single study\",\n      \"pmids\": [\"27374770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CDC45 null mouse embryos fail to develop past implantation; inner cell mass shows impaired proliferation, establishing CDC45 as essential for mammalian post-implantation development and cell proliferation in vivo. Heterozygous mice develop normally, suggesting hemizygosity of CDC45 alone is insufficient to cause cardiac/craniofacial defects in DiGeorge syndrome.\",\n      \"method\": \"Gene targeting (knockout mouse), embryo culture, immunostaining\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — homozygous KO mouse with defined developmental phenotype, single lab\",\n      \"pmids\": [\"11416137\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CDC45 is an essential eukaryotic DNA replication factor that, together with MCM2-7 and GINS, forms the CMG (Cdc45/Mcm2-7/GINS) replicative helicase complex: CDC45 and GINS bridge the MCM2/MCM5 gate to allosterically activate MCM2-7 ATPase and helicase activity, with CDC45 itself guarding the leading-strand template via its RecJ-like fold; CDC45 is loaded at origins in a DDK- and CDK-dependent, stepwise manner (requiring Sld3, Mcm10, TopBP1/Xmus101, and other factors), is rate-limiting for origin firing, travels with the replisome throughout elongation, loads RPA onto nascent ssDNA, bridges replicative polymerases to the helicase, and is targeted by checkpoint kinases (Chk1 displaces it from chromatin; Chk2 directly phosphorylates CMG subunits to inhibit helicase activity), while loss-of-function mutations cause Meier-Gorlin syndrome and craniosynostosis in humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CDC45 is an essential eukaryotic DNA replication factor that, together with MCM2-7 and GINS, constitutes the CMG (Cdc45/Mcm2-7/GINS) replicative helicase [#1, #2]. CDC45 and GINS bridge the Mcm2/Mcm5 gate of the helicase ring to form a topologically closed assembly, and this association elevates MCM2-7 ATP hydrolysis ~100-fold and converts the ring into a processive 3'-to-5' helicase that translocates on the leading-strand template [#2, #3, #11]. CDC45 retains a RecJ-like (DHH-family) fold whose ssDNA-binding groove guards the leading strand within the CMG, and mutations in this fold diminish helicase activity [#4, #12, #13]. CDC45 is loaded at origins in a stepwise, DDK- and CDK-dependent manner downstream of pre-RC assembly, requiring Mcm10, Sld3, TopBP1/Dpb11, and DONSON, with GINS displacing Sld3/Dpb11 to mature the CMS intermediate into the active CMG [#10, #21, #25, #29, #44, #49]; the timing of CDC45 origin association is the key determinant of origin firing time and is rate-limiting for initiation [#24, #45]. Beyond unwinding, CDC45 coordinates polymerase recruitment, tethering DNA polymerase alpha onto MCM via direct binding, supporting sequential loading of RPA, pol alpha, and PCNA, and catalytically loading RPA onto nascent ssDNA [#8, #9, #16, #33]. CDC45 travels with the replisome throughout elongation and acts as a checkpoint hub: Chk2 phosphorylates CMG subunits to inhibit helicase activity, Chk1 displaces CDC45 from chromatin, and the CDC45 disordered loop recruits Rad53/checkpoint kinase to stalled forks [#7, #17, #27, #28]. Biallelic partial loss-of-function mutations in CDC45 cause Meier-Gorlin syndrome and craniosynostosis, and CDC45 is essential for mammalian post-implantation development [#52, #53].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established CDC45 as a genetically required initiation factor acting after START and downstream of pre-RC components, answering where in the cell cycle CDC45 functions.\",\n      \"evidence\": \"Yeast genetics, synthetic lethality with ORC/MCM alleles, 2D gel origin firing analysis, and CDC7/Dbf4 epistasis\",\n      \"pmids\": [\"9001208\", \"9356482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define a biochemical activity for CDC45\", \"Mechanism of CDC45-dependent origin activation unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrated that CDC45 drives the origin-unwinding step and orders the loading of downstream replication factors, distinguishing helicase activation from polymerase recruitment.\",\n      \"evidence\": \"Xenopus egg extract immunodepletion, supercoiling/unwinding assays, chromatin fractionation, and aphidicolin uncoupling\",\n      \"pmids\": [\"10882098\", \"10886370\", \"9755170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the helicase enzyme itself\", \"How CDC45 physically couples to MCM unclear at this stage\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified CDC45-MCM2-7-GINS as a single stable complex (CMG) that is the eukaryotic replicative helicase, resolving the long-standing question of the replicative helicase identity.\",\n      \"evidence\": \"Immunoaffinity purification with MS, in vitro helicase assay on purified Drosophila CMG, and RNAi knockdown\",\n      \"pmids\": [\"16531994\", \"16798881\", \"16483939\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how GINS/CDC45 mechanistically activate MCM\", \"Strand handling within the helicase unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed mechanistically that CDC45 and GINS association activates the MCM ATPase and helicase, quantifying the activation and demonstrating cooperative complex formation.\",\n      \"evidence\": \"Reconstitution from recombinant Drosophila proteins with ATPase, helicase, and pulldown assays\",\n      \"pmids\": [\"20122406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of activation not yet visualized\", \"Did not address leading vs lagging strand routing\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided the structural explanation for CMG activation, showing GINS and CDC45 bridge the Mcm2/Mcm5 gate to close the helicase ring.\",\n      \"evidence\": \"Single-particle EM of MCM2-7 and CMG with functional interpretation\",\n      \"pmids\": [\"21378962\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Resolution insufficient for atomic detail of CDC45\", \"DNA path within channel not directly resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined CDC45 as an ssDNA-binding RecJ/DHH-fold protein that engages 3'-protruding and forked structures, linking its architecture to a strand-handling role.\",\n      \"evidence\": \"Recombinant human/yeast CDC45 with EMSA, AFM, SPR, SAXS binding assays and in vivo mutant phenotypes\",\n      \"pmids\": [\"22147708\", \"24293646\", \"23382391\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Most DHH catalytic residues not conserved; no nuclease activity demonstrated\", \"Single-lab biochemistry\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established that CDC45 guards the leading strand within the CMG, defining its role in strand exclusion during unwinding.\",\n      \"evidence\": \"DNA-protein crosslinking, site-directed mutagenesis, and helicase assays on Drosophila CMG\",\n      \"pmids\": [\"25561522\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological consequence of leading-strand guarding in vivo not fully established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the atomic structure of human CDC45 and connected disease-relevant residues to CMG assembly mechanism.\",\n      \"evidence\": \"X-ray crystallography at 2.1 A with structure-guided mutagenesis and EM integration\",\n      \"pmids\": [\"27189187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational changes upon CMG incorporation not captured\", \"Function of the helical insertion only inferred\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined how DDK phosphorylation of MCM tails templates a two-stage CMG assembly via Cdc45-tail-GINS intermediates, answering how CDC45 recruitment is regulated kinetically.\",\n      \"evidence\": \"Single-molecule fluorescence reconstitution with DDK phosphorylation (building on Sld3/GINS competition and ordered-loading studies)\",\n      \"pmids\": [\"33616038\", \"21362622\", \"25659432\", \"16990792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The inefficient second step's molecular determinants remain undefined\", \"In vivo correspondence of CtG intermediates not directly shown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed CDC45 catalytically loads RPA onto nascent ssDNA, extending its role beyond unwinding to coordinating ssDNA protection at the fork.\",\n      \"evidence\": \"Pull-down, SPR, and real-time RPA-ssDNA binding assays with human recombinant proteins\",\n      \"pmids\": [\"28100698\", \"10518787\", \"17573775\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Catalytic mechanism of RPA handoff not structurally resolved\", \"Single-lab kinetic study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified CDC45 as a checkpoint scaffold recruiting Rad53/checkpoint kinase and linked a CDC45 disordered loop to fork stabilization and a disease mutation.\",\n      \"evidence\": \"Co-IP, in vitro phosphorylation, genetic epistasis, and Meier-Gorlin mutation analysis (with Chk1/Chk2 displacement studies)\",\n      \"pmids\": [\"30595439\", \"22853956\", \"16912045\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How checkpoint engagement at CDC45 is timed relative to elongation unclear\", \"Single-lab Co-IP for several interactions\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked CDC45 dosage to genome stability and established disease causation, showing CDC45 is rate-limiting for origin firing and causative for Meier-Gorlin syndrome.\",\n      \"evidence\": \"DNA fiber and cellular assays under CDC45 overexpression, plus whole-exome sequencing and patient-cell functional validation, with KO mouse lethality\",\n      \"pmids\": [\"26919204\", \"27374770\", \"53\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific basis of MGS/craniosynostosis phenotype unexplained\", \"Quantitative link between CDC45 level and origin number incompletely defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CDC45 abundance, post-translational regulation (PP2A, ubiquitin/APC-C, DNAJA1 chaperone, Myc-driven recruitment) and checkpoint signaling are integrated to set origin firing rates and protect forks across differentiated tissues remains unresolved.\",\n      \"evidence\": \"Not established in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model of CDC45 dosage control in vivo\", \"Direct ubiquitylation and APC/C targeting not biochemically demonstrated\", \"Tissue specificity of regulation unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [13, 14, 15]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [1, 2, 11, 12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8, 9, 16, 33]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 34, 50]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [6, 27, 40]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [32, 50]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [1, 2, 11, 19]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [18, 24, 53]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [17, 27, 28]}\n    ],\n    \"complexes\": [\"CMG helicase (Cdc45/Mcm2-7/GINS)\", \"Replisome progression complex (RPC)\", \"Cdc45-MCM2-7-Sld3 (CMS) intermediate\"],\n    \"partners\": [\"MCM7\", \"GINS (PSF2)\", \"POLA1\", \"RPA\", \"SLD3\", \"TOPBP1\", \"MCM10\", \"DONSON\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}