{"gene":"MCM6","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1999,"finding":"The ATP binding activity of MCM6 within the MCM4-MCM6-MCM7 complex is critical for DNA helicase activity of the complex; mutagenesis of conserved ATPase motifs in MCM6 abolished helicase activity, while MCM4 contributes to single-stranded DNA binding. The helicase and ssDNA binding activities of the complex can be separated.","method":"Expression of mouse MCM4/6/7 in insect cells; Walker A/B ATPase motif mutagenesis; in vitro helicase and ssDNA binding assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic reconstitution with site-directed mutagenesis; multiple orthogonal functional assays in a single focused study","pmids":["10567526"],"is_preprint":false},{"year":1999,"finding":"Human MCM5 and MCM6 gene transcription is regulated by E2F transcription factor binding to multiple E2F sites in their promoters; mutation of E2F sites in the MCM6 promoter abolished serum-stimulated promoter activity, and forced E2F1 expression induced endogenous MCM6 expression.","method":"Promoter mutagenesis, luciferase reporter assays, exogenous E2F1 expression in REF52 cells","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter mutagenesis and reporter assays in one lab with two orthogonal approaches (mutation + forced expression)","pmids":["10327050"],"is_preprint":false},{"year":1999,"finding":"Fission yeast SpMcm6p (encoded by mis5+) associates with chromatin DNA specifically during G1 and S phases, localizes to discrete foci on chromatin at replication origins (ars2004 and ars3002) as shown by chromatin immunoprecipitation, and is loaded onto origins by ORC.","method":"Cell fractionation, immunostaining of spread nuclei, chromatin immunoprecipitation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal ChIP at specific origins, immunostaining, and fractionation; multiple orthogonal methods; fission yeast ortholog (mis5/MCM6)","pmids":["10490657"],"is_preprint":false},{"year":1997,"finding":"Human MCM6 protein (HsMcm6) undergoes cell-cycle-dependent changes in subcellular localization, peaking at G1/S phase; it exists in two forms (detergent-extractable and nucleus-bound) and co-immunoprecipitates with HsMcm2 and HsMcm7 in vivo, forming a heteromeric complex.","method":"Immunoprecipitation, subcellular fractionation with NP-40 extraction, cell cycle synchronization and western blotting","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus fractionation; single lab, two orthogonal methods","pmids":["9286856"],"is_preprint":false},{"year":1998,"finding":"Human MCM6 protein associates with chromatin as part of a multiprotein complex containing other MCM proteins, as demonstrated by micrococcal nuclease digestion releasing MCM6-containing complexes from chromatin; a fraction of MCM6 also occurs as a monomer on chromatin.","method":"Micrococcal nuclease digestion of chromatin, western blotting, co-fractionation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel use of nuclease-based chromatin fractionation; single lab, single primary method","pmids":["9516426"],"is_preprint":false},{"year":2002,"finding":"Mouse Cdt1 directly interacts with MCM6 via the conserved carboxyl-terminal region of Cdt1 (amino acids 407–477); geminin inhibits Cdt1's DNA binding activity (which overlaps with the geminin-binding domain, aa 177–380) but does not directly block the Cdt1–MCM6 interaction domain.","method":"Yeast two-hybrid analysis, bacterial expression and purification of mouse Cdt1, domain-mapping experiments, DNA-binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding assays with purified proteins, domain mapping, and yeast two-hybrid; multiple orthogonal methods in one study","pmids":["12192004"],"is_preprint":false},{"year":2006,"finding":"Phosphorylation of MCM4 at Thr-19 and Thr-110 by CDK2/CDK1 inactivates the MCM4-MCM6-MCM7 helicase complex; EBV protein kinase (EBV-PK) also phosphorylates MCM6 and additional MCM4 sites to further inhibit helicase activity. Introducing N-terminal MCM4 mutations reduced CDK2/cyclin A inhibition but EBV-PK still inhibited both wild-type and mutant complexes, confirming MCM6 phosphorylation contributes.","method":"In vitro kinase assays with CDK2/cyclin A and EBV-PK on MCM4-6-7 hexamer; site-directed mutagenesis of MCM4; helicase activity assays","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and enzymatic activity readout; multiple kinases and mutant comparisons in one study","pmids":["17005684"],"is_preprint":false},{"year":2009,"finding":"Budding yeast Mcm6 directly binds to the checkpoint mediator Mrc1 via the 168-aa C-terminal region of Mcm6 and the conserved coiled-coil region of Mrc1; two amino acid substitutions in Mcm6's C-terminal region abolish this interaction and cause a severe defect in DNA replication checkpoint activation specifically in response to MMS-induced stress (but not HU), and the phenotype is suppressed by fusing Mcm6 directly to Mrc1.","method":"Two-hybrid interaction screen, in vivo co-immunoprecipitation, point mutagenesis, checkpoint assays with MMS and HU, genetic fusion rescue experiment","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, mutagenesis, genetic epistasis (fusion rescue); multiple orthogonal methods, clear mechanistic conclusion","pmids":["19620285"],"is_preprint":false},{"year":2010,"finding":"The Mcm6/2 and Mcm5/3 ATPase active sites (defined by Walker B box and arginine finger motifs) contribute to the function of the putative Mcm2/5 'gate' in the Saccharomyces cerevisiae Mcm2-7 hexameric helicase ring; mutational analysis shows these sites modulate overall Mcm2-7 activity differentially.","method":"Walker B box and arginine finger mutagenesis at individual Mcm2-7 ATPase active sites; in vitro ATPase and helicase activity assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assays with site-directed mutagenesis across multiple active sites; rigorous comparative analysis","pmids":["20484375"],"is_preprint":false},{"year":2010,"finding":"Chromatin-bound MCM6 (Mcm6*) in G1 phase exists in two distinct populations (low and high) separated by the restriction point; cells with high Mcm6* levels reside in late G1 and cycle with committed phases (S, G2, M), as shown by kinetic serum withdrawal and aphidicolin/mimosine/nocodazole experiments.","method":"Flow cytometry with Triton X-100 extraction followed by methanol fixation; antibody staining for Mcm6*, PCNA*, DNA content, and mitotic marker; serum starvation/release kinetics","journal":"BMC cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization/quantification experiment with functional cell cycle consequence; multiple cell lines tested, single lab","pmids":["20398392"],"is_preprint":false},{"year":2021,"finding":"BLM helicase directly and physically interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase (a third site mediates binding after DNA damage). Disruption of the BLM–Mcm6 S-phase interaction leads to supra-normal DNA replication speed in unperturbed cells (requiring BLM helicase activity), delayed repair of replication-dependent DSBs, and hypersensitivity to DNA damage and replication stress.","method":"Identification of BLM complex composition during S-phase, direct binding assays with distinct Mcm6 domains, cell-based replication speed assays, DNA damage repair assays, BLM helicase mutant analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct physical interaction mapping with domain-specific binding sites, functional phenotypic readouts (replication speed, DSB repair), helicase activity requirement demonstrated; multiple orthogonal methods","pmids":["34370039"],"is_preprint":false},{"year":2010,"finding":"NMR chemical shift assignments of the Cdt1-binding domain (CBD) of human MCM6 show it adopts a 'winged-helix' fold typical of protein–nucleic acid interaction domains; this domain directly mediates the Cdt1–MCM6 interaction for chromatin loading of the MCM2-7 complex.","method":"Triple-resonance NMR spectroscopy; chemical shift assignments deposited (BMRB 16396)","journal":"Biomolecular NMR assignments","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structural characterization of the CBD domain; single lab, assignments only (no full structure or mutagenesis in this paper)","pmids":["20623209"],"is_preprint":false},{"year":2024,"finding":"SIRT7-mediated lysine crotonylation of MCM6 at K599 (MCM6-K599cr) is upregulated in response to DNA replication stress; this crotonylation is associated with disassembly of the MCM2-7 complex and is regulated by RNF8-mediated ubiquitination of MCM6.","method":"Western blotting for Kcr marks, MCM6 knockdown proliferation/DNA replication assays, identification of K599 crotonylation site, RNF8 co-expression ubiquitination assays, kaempferol treatment as SIRT7 regulator","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific PTM identification with upstream writer (SIRT7) and regulatory ubiquitin E3 (RNF8) identified; single lab, multiple methods","pmids":["39477811"],"is_preprint":false},{"year":2024,"finding":"RNF125 E3 ubiquitin ligase interacts with MCM6 and mediates its ubiquitination; co-transfection experiments show RNF125 promotes HCC cell proliferation mainly through MCM6.","method":"Pull-down assay, co-immunoprecipitation, ubiquitination assay, co-transfection with MCM6 rescue","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, pull-down, and ubiquitination assay with functional rescue; single lab, three orthogonal methods","pmids":["38298426"],"is_preprint":false},{"year":2024,"finding":"Avermectin B1a binds to the CDT1-binding domain (CBD) of MCM6 (at residues Glu763, Ile760, Arg771, Glu774), blocking the MCM6–CDT1 interaction and thereby inhibiting DNA replication licensing, causing G0/G1 cell cycle arrest and apoptosis; overexpression of MCM6 or CDT1 reverses these cytotoxic effects.","method":"Molecular docking, modified ELISA-based binding assay, cell cycle analysis, viability assays, MCM6/CDT1 overexpression rescue","journal":"Environmental pollution","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding confirmed by modified ELISA plus molecular docking; rescue experiments with overexpression; single lab","pmids":["39579921"],"is_preprint":false},{"year":2022,"finding":"MCM6 is a direct transcriptional target of the YAP-TEAD complex in gastric cancer; the YAP-TEAD complex binds to the MCM6 promoter to induce transcription. MCM6 in turn activates PI3K/Akt/GSK3β signaling. MCM6 deficiency sensitizes cells to chemo/radiotherapy by causing DNA breaks and blocking ATR/Chk1-mediated DNA damage response.","method":"RNA sequencing, microarray, chromatin immunoprecipitation PCR (YAP-TEAD binding to MCM6 promoter), luciferase reporter assays, MCM6 knockdown in organoids and xenografts, western blotting for PI3K/Akt/GSK3β and ATR/Chk1 pathway components","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP-PCR confirms direct promoter binding, luciferase validates transcriptional activation, multiple downstream pathway readouts, in vivo validation; multiple orthogonal methods in one study","pmids":["36185598"],"is_preprint":false},{"year":2021,"finding":"MCM6 knockdown in HCC cells promotes EMT and activates MEK/ERK signaling (decreased pERK, decreased EMT markers upon knockdown); MCM6 expression also promotes MEK/ERK-driven migration and invasion in vivo.","method":"Western blotting for ERK pathway and EMT markers, immunofluorescence staining, subcutaneous and orthotopic xenograft models, wound healing, Transwell assays","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pathway activation confirmed by western blotting and in vivo models; single lab, multiple readouts but no direct binding or reconstitution","pmids":["29357919"],"is_preprint":false},{"year":2018,"finding":"MCM6 knockdown in HCC cells causes a delay in S/G2-phase progression accompanied by downregulation of CDK2, CDK4, CyclinA, CyclinB1, CyclinD1, and CyclinE.","method":"siRNA knockdown, flow cytometry cell cycle analysis, western blotting for cyclins and CDKs","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — KD with defined cell cycle phenotype and molecular markers; single lab, single method set","pmids":["29463213"],"is_preprint":false},{"year":2023,"finding":"MCM6 promotes ICC (intrahepatic cholangiocarcinoma) progression by upregulating E2F1, which mediates EMT; E2F1 knockdown partially blocked the pro-malignant effects of MCM6 overexpression, placing MCM6 upstream of E2F1 in this pathway.","method":"GSEA, MCM6 knockdown and overexpression, western blotting and functional assays, E2F1 rescue knockdown experiments, in vivo xenograft","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — genetic epistasis (E2F1 KD rescues MCM6 OE), functional assays; single lab","pmids":["37185675"],"is_preprint":false},{"year":2025,"finding":"MCM6 promotes cisplatin resistance in bladder cancer by enhancing DNA damage repair (DDR); MCM6 knockdown reduced nuclear c-Myc levels and promoted its ubiquitin-mediated degradation, increasing DNA damage. Conversely, c-Myc (as a transcription factor) binds the MCM6 promoter to drive MCM6 transcription, forming a positive feedback loop.","method":"MCM6 knockdown/overexpression, western blotting for DDR markers and c-Myc, ubiquitination assays, chromatin immunoprecipitation for c-Myc binding to MCM6 promoter, in vivo xenograft cisplatin sensitivity","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ChIP confirms c-Myc binding to MCM6 promoter; ubiquitination and DDR assays; single lab, mechanistic loop proposed with experimental support","pmids":["39805445"],"is_preprint":false},{"year":2023,"finding":"MCM6 interacts with E6AP (UBE3A) ubiquitin ligase (identified by yeast two-hybrid); however, E6AP knockout enhanced ubiquitination of MCM2/4/6, indicating E6AP is not the E3 ubiquitin ligase for these MCM proteins. Ablation of both MCM6 and E6AP synergistically suppressed proliferation and migration of lung adenocarcinoma cells in vitro and in vivo.","method":"Yeast two-hybrid assay (MCM6–E6AP interaction), ubiquitination assays upon E6AP KO, proliferation/migration assays, nude mouse xenograft models","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — yeast two-hybrid plus ubiquitination assay; negative result (E6AP is not the E3 for MCM6) and synergistic functional readout; single lab","pmids":["37454373"],"is_preprint":false},{"year":2025,"finding":"Emodepside directly binds to the CDT1-binding domain (CBD) of MCM6 at residues Lys754, Ile760, and Lys770, competitively blocking the MCM6–CDT1 interaction, inhibiting DNA replication licensing, causing G0/G1 cell cycle arrest and apoptosis in human corneal stromal cells.","method":"Modified ELISA binding assay, drug affinity responsive target stability (DARTS) assay, molecular docking, EdU incorporation assay, cell cycle and apoptosis analysis","journal":"Toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct binding confirmed by DARTS and modified ELISA; functional cell cycle and DNA synthesis readouts; single lab, consistent with prior avermectin study","pmids":["41203057"],"is_preprint":false},{"year":2023,"finding":"De novo MCM6 missense variant p.(Cys158Tyr), affecting a zinc-binding cysteine in the MCM6 zinc finger signature, causes defects in both ciliogenesis and cell proliferation in patient-derived fibroblasts, consistent with an essential role of the zinc-binding domain in MCM-complex dimerization and helicase activity induction.","method":"Trio exome/genome sequencing, patient-derived fibroblast functional assays for ciliogenesis and cell proliferation","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function patient variant with defined structural domain, fibroblast functional assays for two orthogonal readouts; two unrelated patients with identical variant","pmids":["37198333"],"is_preprint":false},{"year":2024,"finding":"MCM6 inhibits decidualization of human endometrial stromal cells; MCM6 overexpression promotes G1/S transition and restores proliferation inhibited by E2+P4 treatment via aberrant ERK activation; treatment with ERK agonist Ro 67-7476 restores MCM6 expression, revealing a MCM6/ERK feedback loop that negatively regulates decidualization.","method":"In vitro decidualization model with E2+P4, MCM6 overexpression, flow cytometry cell cycle analysis, western blotting for ERK pathway, ERK agonist treatment","journal":"Reproductive sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, overexpression + pharmacological intervention, no direct biochemical binding assay; mechanistic pathway placement is correlative","pmids":["38347378"],"is_preprint":false}],"current_model":"MCM6 is a core subunit of the heterohexameric MCM2-7 replicative helicase whose ATP-binding activity is essential for the DNA unwinding activity of the MCM4-MCM6-MCM7 sub-complex; it is loaded onto replication origins during G1 phase via direct interaction with Cdt1 (through its C-terminal winged-helix CDT1-binding domain), is regulated by CDK/EBV-PK-mediated phosphorylation of MCM4 that inactivates the helicase, interacts with the checkpoint mediator Mrc1/Claspin to sense MMS-induced replication stress, and binds the BLM helicase in a cell-cycle-phase-specific manner to restrain replication speed; additionally, MCM6 transcription is driven by E2F and (in cancer contexts) by YAP-TEAD and c-Myc, while its protein stability is regulated by RNF125- and RNF8-mediated ubiquitination and by SIRT7-mediated crotonylation at K599 during replication stress."},"narrative":{"mechanistic_narrative":"MCM6 is a core subunit of the heterohexameric MCM2-7 replicative helicase that drives origin licensing and DNA unwinding during the G1/S transition [PMID:10567526, PMID:9286856, PMID:20484375]. Within the MCM4-MCM6-MCM7 sub-complex, the ATP-binding activity of MCM6 is critical for helicase function: mutagenesis of its conserved Walker A/B ATPase motifs abolishes unwinding, and its ATPase active site (with MCM2) contributes to the Mcm2/5 'gate' that gates the hexameric ring, while ssDNA binding maps to MCM4 [PMID:10567526, PMID:20484375]. MCM6 is loaded onto chromatin at replication origins during G1 phase in an ORC-dependent manner, where it associates with other MCM subunits and exists in both chromatin-bound and soluble forms across the cell cycle [PMID:10490657, PMID:9286856, PMID:9516426, PMID:20398392]. Origin licensing is mediated by a direct interaction between Cdt1 and the C-terminal Cdt1-binding domain (CBD) of MCM6, a domain that adopts a winged-helix fold; small molecules that occupy the CBD competitively block the MCM6-CDT1 interaction, arresting cells in G0/G1 [PMID:12192004, PMID:20623209, PMID:39579921, PMID:41203057]. Helicase activity is restrained by CDK- and EBV-PK-mediated phosphorylation, and MCM6 additionally couples replication to genome surveillance by binding the checkpoint mediator Mrc1/Claspin to activate the MMS-induced replication checkpoint and by binding the BLM helicase in a cell-cycle-phase-specific manner to limit replication fork speed [PMID:17005684, PMID:19620285, PMID:34370039]. MCM6 abundance is controlled transcriptionally by E2F and, in cancer, by YAP-TEAD and c-Myc, and post-translationally by ubiquitination (RNF125, RNF8) and SIRT7-mediated K599 crotonylation that accompanies MCM2-7 disassembly under replication stress [PMID:10327050, PMID:39477811, PMID:38298426, PMID:36185598, PMID:39805445]. A de novo missense variant disrupting a zinc-binding cysteine of the MCM6 zinc finger impairs ciliogenesis and proliferation in patient fibroblasts, linking MCM6 dysfunction to human disease [PMID:37198333].","teleology":[{"year":1997,"claim":"Established that human MCM6 is a physical component of a heteromeric MCM complex and that its localization is cell-cycle regulated, peaking at G1/S, defining it as a replication-associated factor.","evidence":"Co-immunoprecipitation with MCM2/MCM7 and subcellular fractionation across synchronized cells","pmids":["9286856"],"confidence":"Medium","gaps":["Did not resolve the stoichiometry or which subunits contact MCM6 directly","No functional readout of the complex"]},{"year":1999,"claim":"Defined the enzymatic core of MCM6 by showing its ATP-binding activity is essential for helicase function of the MCM4-6-7 sub-complex, separating helicase from ssDNA-binding activities.","evidence":"Recombinant mouse MCM4/6/7 with Walker A/B motif mutagenesis and in vitro helicase and ssDNA-binding assays","pmids":["10567526"],"confidence":"High","gaps":["MCM4-6-7 sub-complex is not the physiological MCM2-7 ring","Did not address how ATP hydrolysis is coupled to translocation"]},{"year":1999,"claim":"Connected MCM6 expression to cell-cycle entry by showing E2F drives its transcription, explaining serum-responsive induction of licensing capacity.","evidence":"Promoter mutagenesis, luciferase reporters and forced E2F1 expression in REF52 cells","pmids":["10327050"],"confidence":"Medium","gaps":["Did not establish which endogenous E2F member dominates in vivo","No link to protein-level licensing output"]},{"year":1999,"claim":"Showed origin loading of MCM6 in vivo by mapping the fission yeast ortholog to specific replication origins in an ORC-dependent, G1/S-restricted manner.","evidence":"Cell fractionation, spread-nuclei immunostaining and ChIP at ars2004/ars3002 in S. pombe","pmids":["10490657"],"confidence":"High","gaps":["Ortholog system; human origin engagement inferred","Loading mechanism beyond ORC not resolved here"]},{"year":2002,"claim":"Identified the direct licensing contact by mapping a Cdt1-MCM6 interaction through Cdt1's C-terminal region, distinguishing it from the geminin-regulated DNA-binding domain.","evidence":"Yeast two-hybrid, purified-protein binding and domain mapping of mouse Cdt1","pmids":["12192004"],"confidence":"High","gaps":["Did not localize the reciprocal MCM6 binding surface in this study","Geminin's effect on loading kinetics not measured"]},{"year":2006,"claim":"Defined phosphoregulation of the helicase, showing CDK and EBV-PK phosphorylation (including of MCM6) inactivates the MCM4-6-7 complex.","evidence":"In vitro kinase assays with CDK2/cyclin A and EBV-PK plus MCM4 mutagenesis and helicase readouts","pmids":["17005684"],"confidence":"High","gaps":["MCM6 phosphosites not precisely mapped","Physiological relevance in the MCM2-7 ring not tested"]},{"year":2009,"claim":"Revealed a checkpoint-sensing role by mapping a direct Mcm6 C-terminus to Mrc1 coiled-coil interaction required specifically for the MMS-induced replication checkpoint.","evidence":"Two-hybrid, reciprocal Co-IP, point mutagenesis and fusion-rescue genetics in budding yeast","pmids":["19620285"],"confidence":"High","gaps":["HU-induced checkpoint is independent of this interaction","Human Claspin equivalence not directly tested"]},{"year":2010,"claim":"Refined the catalytic architecture by showing the Mcm6/2 ATPase active site contributes to the Mcm2/5 gate that modulates Mcm2-7 helicase activity.","evidence":"Walker B and arginine-finger mutagenesis across individual active sites with in vitro ATPase/helicase assays in S. cerevisiae","pmids":["20484375"],"confidence":"High","gaps":["Gate opening dynamics not directly visualized","Coupling to DNA loading versus unwinding not separated"]},{"year":2010,"claim":"Linked chromatin-bound MCM6 levels to commitment past the restriction point, defining distinct G1 populations with different cycling fates.","evidence":"Flow cytometry with detergent extraction and serum-withdrawal/drug arrest kinetics across cell lines","pmids":["20398392"],"confidence":"Medium","gaps":["Correlative rather than causal link to restriction-point control","Molecular trigger for the high-Mcm6* state unknown"]},{"year":2010,"claim":"Provided structural identity for the licensing module by showing the MCM6 CBD adopts a winged-helix fold.","evidence":"Triple-resonance NMR chemical-shift assignments of human MCM6 CBD (BMRB 16396)","pmids":["20623209"],"confidence":"Medium","gaps":["Assignments only; no full structure or Cdt1-bound complex","No mutagenesis of the binding interface in this work"]},{"year":2021,"claim":"Uncovered a fork-speed control mechanism via phase-specific BLM-Mcm6 interactions, with the S-phase contact restraining replication speed and protecting genome integrity.","evidence":"BLM complex composition, domain-specific binding to N-/C-terminal Mcm6, replication speed and DSB-repair assays with BLM helicase mutants","pmids":["34370039"],"confidence":"High","gaps":["Switch mechanism between N-terminal and CBD sites not resolved","How accelerated forks cause DSBs mechanistically unclear"]},{"year":2023,"claim":"Linked MCM6 to human Mendelian disease by showing a de novo zinc-finger cysteine variant impairs ciliogenesis and proliferation.","evidence":"Trio exome/genome sequencing and patient-derived fibroblast functional assays","pmids":["37198333"],"confidence":"Medium","gaps":["Helicase-level consequence of the variant not directly measured","Mechanistic basis of the ciliary phenotype unresolved"]},{"year":2024,"claim":"Defined PTM-based regulation of complex stability by identifying SIRT7-mediated K599 crotonylation, coupled to RNF8 ubiquitination, that accompanies MCM2-7 disassembly under replication stress.","evidence":"Site mapping, Kcr western blotting, knockdown replication assays and RNF8 ubiquitination assays","pmids":["39477811"],"confidence":"Medium","gaps":["Causal order of crotonylation versus ubiquitination not established","Direct effect of K599cr on helicase activity not measured"]},{"year":2024,"claim":"Identified RNF125 as an E3 ligase that ubiquitinates MCM6 and drives proliferation through it in hepatocellular carcinoma.","evidence":"Pull-down, Co-IP, ubiquitination assay and MCM6 rescue co-transfection","pmids":["38298426"],"confidence":"Medium","gaps":["Whether ubiquitination is degradative or signaling not resolved","Lysine target sites not mapped"]},{"year":2022,"claim":"Placed MCM6 in oncogenic transcriptional circuitry as a direct YAP-TEAD target that feeds PI3K/Akt signaling and supports the ATR/Chk1 damage response.","evidence":"RNA-seq, ChIP-PCR, luciferase reporters, organoid/xenograft knockdown and pathway western blots in gastric cancer","pmids":["36185598"],"confidence":"High","gaps":["Whether the PI3K/Akt link is direct or replication-dependent unclear","Mechanism connecting MCM6 loss to ATR/Chk1 failure not detailed"]},{"year":2025,"claim":"Established a c-Myc/MCM6 positive feedback loop supporting DNA damage repair and cisplatin resistance.","evidence":"Knockdown/overexpression, ChIP for c-Myc on MCM6 promoter, ubiquitination and DDR assays, xenograft chemosensitivity","pmids":["39805445"],"confidence":"Medium","gaps":["Direct biochemical basis of MCM6-promoted c-Myc stabilization unclear","Single tumor context"]},{"year":2025,"claim":"Demonstrated druggability of the MCM6 CBD with multiple small molecules that competitively block CDT1 binding and arrest cells, with MCM6/CDT1 overexpression rescuing toxicity.","evidence":"Modified ELISA, DARTS, molecular docking and cell-cycle/apoptosis assays with avermectin B1a and emodepside","pmids":["39579921","41203057"],"confidence":"Medium","gaps":["Residue-level binding from docking not all confirmed structurally","Selectivity over other winged-helix domains untested"]},{"year":null,"claim":"How the diverse cancer-context signaling roles (ERK/EMT, E2F1, decidualization) mechanistically derive from the core licensing/helicase function of MCM6 remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No direct biochemical link between MCM6 helicase activity and ERK/EMT outputs","Whether non-replication roles reflect off-complex MCM6 is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0,8]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[2,4,9]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0,5,8]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,9,17]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[7,10]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[7,12]}],"complexes":["MCM2-7 helicase","MCM4-MCM6-MCM7 sub-complex"],"partners":["MCM2","MCM7","MCM4","CDT1","MRC1","BLM","RNF8","RNF125"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14566","full_name":"DNA replication licensing factor MCM6","aliases":["p105MCM"],"length_aa":821,"mass_kda":92.9,"function":"Acts as a component of the MCM2-7 complex (MCM complex) which is the replicative helicase essential for 'once per cell cycle' DNA replication initiation and elongation in eukaryotic cells. Core component of CDC45-MCM-GINS (CMG) helicase, the molecular machine that unwinds template DNA during replication, and around which the replisome is built (PubMed:16899510, PubMed:32453425, PubMed:34694004, PubMed:34700328, PubMed:35585232, PubMed:9305914). The active ATPase sites in the MCM2-7 ring are formed through the interaction surfaces of two neighboring subunits such that a critical structure of a conserved arginine finger motif is provided in trans relative to the ATP-binding site of the Walker A box of the adjacent subunit. The six ATPase active sites, however, are likely to contribute differentially to the complex helicase activity (PubMed:32453425)","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q14566/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MCM6","classification":"Common 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The helicase and ssDNA binding activities of the complex can be separated.\",\n      \"method\": \"Expression of mouse MCM4/6/7 in insect cells; Walker A/B ATPase motif mutagenesis; in vitro helicase and ssDNA binding assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic reconstitution with site-directed mutagenesis; multiple orthogonal functional assays in a single focused study\",\n      \"pmids\": [\"10567526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human MCM5 and MCM6 gene transcription is regulated by E2F transcription factor binding to multiple E2F sites in their promoters; mutation of E2F sites in the MCM6 promoter abolished serum-stimulated promoter activity, and forced E2F1 expression induced endogenous MCM6 expression.\",\n      \"method\": \"Promoter mutagenesis, luciferase reporter assays, exogenous E2F1 expression in REF52 cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter mutagenesis and reporter assays in one lab with two orthogonal approaches (mutation + forced expression)\",\n      \"pmids\": [\"10327050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Fission yeast SpMcm6p (encoded by mis5+) associates with chromatin DNA specifically during G1 and S phases, localizes to discrete foci on chromatin at replication origins (ars2004 and ars3002) as shown by chromatin immunoprecipitation, and is loaded onto origins by ORC.\",\n      \"method\": \"Cell fractionation, immunostaining of spread nuclei, chromatin immunoprecipitation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal ChIP at specific origins, immunostaining, and fractionation; multiple orthogonal methods; fission yeast ortholog (mis5/MCM6)\",\n      \"pmids\": [\"10490657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Human MCM6 protein (HsMcm6) undergoes cell-cycle-dependent changes in subcellular localization, peaking at G1/S phase; it exists in two forms (detergent-extractable and nucleus-bound) and co-immunoprecipitates with HsMcm2 and HsMcm7 in vivo, forming a heteromeric complex.\",\n      \"method\": \"Immunoprecipitation, subcellular fractionation with NP-40 extraction, cell cycle synchronization and western blotting\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus fractionation; single lab, two orthogonal methods\",\n      \"pmids\": [\"9286856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human MCM6 protein associates with chromatin as part of a multiprotein complex containing other MCM proteins, as demonstrated by micrococcal nuclease digestion releasing MCM6-containing complexes from chromatin; a fraction of MCM6 also occurs as a monomer on chromatin.\",\n      \"method\": \"Micrococcal nuclease digestion of chromatin, western blotting, co-fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel use of nuclease-based chromatin fractionation; single lab, single primary method\",\n      \"pmids\": [\"9516426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Mouse Cdt1 directly interacts with MCM6 via the conserved carboxyl-terminal region of Cdt1 (amino acids 407–477); geminin inhibits Cdt1's DNA binding activity (which overlaps with the geminin-binding domain, aa 177–380) but does not directly block the Cdt1–MCM6 interaction domain.\",\n      \"method\": \"Yeast two-hybrid analysis, bacterial expression and purification of mouse Cdt1, domain-mapping experiments, DNA-binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assays with purified proteins, domain mapping, and yeast two-hybrid; multiple orthogonal methods in one study\",\n      \"pmids\": [\"12192004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Phosphorylation of MCM4 at Thr-19 and Thr-110 by CDK2/CDK1 inactivates the MCM4-MCM6-MCM7 helicase complex; EBV protein kinase (EBV-PK) also phosphorylates MCM6 and additional MCM4 sites to further inhibit helicase activity. Introducing N-terminal MCM4 mutations reduced CDK2/cyclin A inhibition but EBV-PK still inhibited both wild-type and mutant complexes, confirming MCM6 phosphorylation contributes.\",\n      \"method\": \"In vitro kinase assays with CDK2/cyclin A and EBV-PK on MCM4-6-7 hexamer; site-directed mutagenesis of MCM4; helicase activity assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and enzymatic activity readout; multiple kinases and mutant comparisons in one study\",\n      \"pmids\": [\"17005684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Budding yeast Mcm6 directly binds to the checkpoint mediator Mrc1 via the 168-aa C-terminal region of Mcm6 and the conserved coiled-coil region of Mrc1; two amino acid substitutions in Mcm6's C-terminal region abolish this interaction and cause a severe defect in DNA replication checkpoint activation specifically in response to MMS-induced stress (but not HU), and the phenotype is suppressed by fusing Mcm6 directly to Mrc1.\",\n      \"method\": \"Two-hybrid interaction screen, in vivo co-immunoprecipitation, point mutagenesis, checkpoint assays with MMS and HU, genetic fusion rescue experiment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, mutagenesis, genetic epistasis (fusion rescue); multiple orthogonal methods, clear mechanistic conclusion\",\n      \"pmids\": [\"19620285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The Mcm6/2 and Mcm5/3 ATPase active sites (defined by Walker B box and arginine finger motifs) contribute to the function of the putative Mcm2/5 'gate' in the Saccharomyces cerevisiae Mcm2-7 hexameric helicase ring; mutational analysis shows these sites modulate overall Mcm2-7 activity differentially.\",\n      \"method\": \"Walker B box and arginine finger mutagenesis at individual Mcm2-7 ATPase active sites; in vitro ATPase and helicase activity assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assays with site-directed mutagenesis across multiple active sites; rigorous comparative analysis\",\n      \"pmids\": [\"20484375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Chromatin-bound MCM6 (Mcm6*) in G1 phase exists in two distinct populations (low and high) separated by the restriction point; cells with high Mcm6* levels reside in late G1 and cycle with committed phases (S, G2, M), as shown by kinetic serum withdrawal and aphidicolin/mimosine/nocodazole experiments.\",\n      \"method\": \"Flow cytometry with Triton X-100 extraction followed by methanol fixation; antibody staining for Mcm6*, PCNA*, DNA content, and mitotic marker; serum starvation/release kinetics\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization/quantification experiment with functional cell cycle consequence; multiple cell lines tested, single lab\",\n      \"pmids\": [\"20398392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BLM helicase directly and physically interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase (a third site mediates binding after DNA damage). Disruption of the BLM–Mcm6 S-phase interaction leads to supra-normal DNA replication speed in unperturbed cells (requiring BLM helicase activity), delayed repair of replication-dependent DSBs, and hypersensitivity to DNA damage and replication stress.\",\n      \"method\": \"Identification of BLM complex composition during S-phase, direct binding assays with distinct Mcm6 domains, cell-based replication speed assays, DNA damage repair assays, BLM helicase mutant analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct physical interaction mapping with domain-specific binding sites, functional phenotypic readouts (replication speed, DSB repair), helicase activity requirement demonstrated; multiple orthogonal methods\",\n      \"pmids\": [\"34370039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NMR chemical shift assignments of the Cdt1-binding domain (CBD) of human MCM6 show it adopts a 'winged-helix' fold typical of protein–nucleic acid interaction domains; this domain directly mediates the Cdt1–MCM6 interaction for chromatin loading of the MCM2-7 complex.\",\n      \"method\": \"Triple-resonance NMR spectroscopy; chemical shift assignments deposited (BMRB 16396)\",\n      \"journal\": \"Biomolecular NMR assignments\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structural characterization of the CBD domain; single lab, assignments only (no full structure or mutagenesis in this paper)\",\n      \"pmids\": [\"20623209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SIRT7-mediated lysine crotonylation of MCM6 at K599 (MCM6-K599cr) is upregulated in response to DNA replication stress; this crotonylation is associated with disassembly of the MCM2-7 complex and is regulated by RNF8-mediated ubiquitination of MCM6.\",\n      \"method\": \"Western blotting for Kcr marks, MCM6 knockdown proliferation/DNA replication assays, identification of K599 crotonylation site, RNF8 co-expression ubiquitination assays, kaempferol treatment as SIRT7 regulator\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific PTM identification with upstream writer (SIRT7) and regulatory ubiquitin E3 (RNF8) identified; single lab, multiple methods\",\n      \"pmids\": [\"39477811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF125 E3 ubiquitin ligase interacts with MCM6 and mediates its ubiquitination; co-transfection experiments show RNF125 promotes HCC cell proliferation mainly through MCM6.\",\n      \"method\": \"Pull-down assay, co-immunoprecipitation, ubiquitination assay, co-transfection with MCM6 rescue\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, pull-down, and ubiquitination assay with functional rescue; single lab, three orthogonal methods\",\n      \"pmids\": [\"38298426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Avermectin B1a binds to the CDT1-binding domain (CBD) of MCM6 (at residues Glu763, Ile760, Arg771, Glu774), blocking the MCM6–CDT1 interaction and thereby inhibiting DNA replication licensing, causing G0/G1 cell cycle arrest and apoptosis; overexpression of MCM6 or CDT1 reverses these cytotoxic effects.\",\n      \"method\": \"Molecular docking, modified ELISA-based binding assay, cell cycle analysis, viability assays, MCM6/CDT1 overexpression rescue\",\n      \"journal\": \"Environmental pollution\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding confirmed by modified ELISA plus molecular docking; rescue experiments with overexpression; single lab\",\n      \"pmids\": [\"39579921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MCM6 is a direct transcriptional target of the YAP-TEAD complex in gastric cancer; the YAP-TEAD complex binds to the MCM6 promoter to induce transcription. MCM6 in turn activates PI3K/Akt/GSK3β signaling. MCM6 deficiency sensitizes cells to chemo/radiotherapy by causing DNA breaks and blocking ATR/Chk1-mediated DNA damage response.\",\n      \"method\": \"RNA sequencing, microarray, chromatin immunoprecipitation PCR (YAP-TEAD binding to MCM6 promoter), luciferase reporter assays, MCM6 knockdown in organoids and xenografts, western blotting for PI3K/Akt/GSK3β and ATR/Chk1 pathway components\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-PCR confirms direct promoter binding, luciferase validates transcriptional activation, multiple downstream pathway readouts, in vivo validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"36185598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MCM6 knockdown in HCC cells promotes EMT and activates MEK/ERK signaling (decreased pERK, decreased EMT markers upon knockdown); MCM6 expression also promotes MEK/ERK-driven migration and invasion in vivo.\",\n      \"method\": \"Western blotting for ERK pathway and EMT markers, immunofluorescence staining, subcutaneous and orthotopic xenograft models, wound healing, Transwell assays\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pathway activation confirmed by western blotting and in vivo models; single lab, multiple readouts but no direct binding or reconstitution\",\n      \"pmids\": [\"29357919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MCM6 knockdown in HCC cells causes a delay in S/G2-phase progression accompanied by downregulation of CDK2, CDK4, CyclinA, CyclinB1, CyclinD1, and CyclinE.\",\n      \"method\": \"siRNA knockdown, flow cytometry cell cycle analysis, western blotting for cyclins and CDKs\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — KD with defined cell cycle phenotype and molecular markers; single lab, single method set\",\n      \"pmids\": [\"29463213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MCM6 promotes ICC (intrahepatic cholangiocarcinoma) progression by upregulating E2F1, which mediates EMT; E2F1 knockdown partially blocked the pro-malignant effects of MCM6 overexpression, placing MCM6 upstream of E2F1 in this pathway.\",\n      \"method\": \"GSEA, MCM6 knockdown and overexpression, western blotting and functional assays, E2F1 rescue knockdown experiments, in vivo xenograft\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — genetic epistasis (E2F1 KD rescues MCM6 OE), functional assays; single lab\",\n      \"pmids\": [\"37185675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MCM6 promotes cisplatin resistance in bladder cancer by enhancing DNA damage repair (DDR); MCM6 knockdown reduced nuclear c-Myc levels and promoted its ubiquitin-mediated degradation, increasing DNA damage. Conversely, c-Myc (as a transcription factor) binds the MCM6 promoter to drive MCM6 transcription, forming a positive feedback loop.\",\n      \"method\": \"MCM6 knockdown/overexpression, western blotting for DDR markers and c-Myc, ubiquitination assays, chromatin immunoprecipitation for c-Myc binding to MCM6 promoter, in vivo xenograft cisplatin sensitivity\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ChIP confirms c-Myc binding to MCM6 promoter; ubiquitination and DDR assays; single lab, mechanistic loop proposed with experimental support\",\n      \"pmids\": [\"39805445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MCM6 interacts with E6AP (UBE3A) ubiquitin ligase (identified by yeast two-hybrid); however, E6AP knockout enhanced ubiquitination of MCM2/4/6, indicating E6AP is not the E3 ubiquitin ligase for these MCM proteins. Ablation of both MCM6 and E6AP synergistically suppressed proliferation and migration of lung adenocarcinoma cells in vitro and in vivo.\",\n      \"method\": \"Yeast two-hybrid assay (MCM6–E6AP interaction), ubiquitination assays upon E6AP KO, proliferation/migration assays, nude mouse xenograft models\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — yeast two-hybrid plus ubiquitination assay; negative result (E6AP is not the E3 for MCM6) and synergistic functional readout; single lab\",\n      \"pmids\": [\"37454373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Emodepside directly binds to the CDT1-binding domain (CBD) of MCM6 at residues Lys754, Ile760, and Lys770, competitively blocking the MCM6–CDT1 interaction, inhibiting DNA replication licensing, causing G0/G1 cell cycle arrest and apoptosis in human corneal stromal cells.\",\n      \"method\": \"Modified ELISA binding assay, drug affinity responsive target stability (DARTS) assay, molecular docking, EdU incorporation assay, cell cycle and apoptosis analysis\",\n      \"journal\": \"Toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct binding confirmed by DARTS and modified ELISA; functional cell cycle and DNA synthesis readouts; single lab, consistent with prior avermectin study\",\n      \"pmids\": [\"41203057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"De novo MCM6 missense variant p.(Cys158Tyr), affecting a zinc-binding cysteine in the MCM6 zinc finger signature, causes defects in both ciliogenesis and cell proliferation in patient-derived fibroblasts, consistent with an essential role of the zinc-binding domain in MCM-complex dimerization and helicase activity induction.\",\n      \"method\": \"Trio exome/genome sequencing, patient-derived fibroblast functional assays for ciliogenesis and cell proliferation\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function patient variant with defined structural domain, fibroblast functional assays for two orthogonal readouts; two unrelated patients with identical variant\",\n      \"pmids\": [\"37198333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MCM6 inhibits decidualization of human endometrial stromal cells; MCM6 overexpression promotes G1/S transition and restores proliferation inhibited by E2+P4 treatment via aberrant ERK activation; treatment with ERK agonist Ro 67-7476 restores MCM6 expression, revealing a MCM6/ERK feedback loop that negatively regulates decidualization.\",\n      \"method\": \"In vitro decidualization model with E2+P4, MCM6 overexpression, flow cytometry cell cycle analysis, western blotting for ERK pathway, ERK agonist treatment\",\n      \"journal\": \"Reproductive sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, overexpression + pharmacological intervention, no direct biochemical binding assay; mechanistic pathway placement is correlative\",\n      \"pmids\": [\"38347378\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MCM6 is a core subunit of the heterohexameric MCM2-7 replicative helicase whose ATP-binding activity is essential for the DNA unwinding activity of the MCM4-MCM6-MCM7 sub-complex; it is loaded onto replication origins during G1 phase via direct interaction with Cdt1 (through its C-terminal winged-helix CDT1-binding domain), is regulated by CDK/EBV-PK-mediated phosphorylation of MCM4 that inactivates the helicase, interacts with the checkpoint mediator Mrc1/Claspin to sense MMS-induced replication stress, and binds the BLM helicase in a cell-cycle-phase-specific manner to restrain replication speed; additionally, MCM6 transcription is driven by E2F and (in cancer contexts) by YAP-TEAD and c-Myc, while its protein stability is regulated by RNF125- and RNF8-mediated ubiquitination and by SIRT7-mediated crotonylation at K599 during replication stress.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MCM6 is a core subunit of the heterohexameric MCM2-7 replicative helicase that drives origin licensing and DNA unwinding during the G1/S transition [#0, #3, #8]. Within the MCM4-MCM6-MCM7 sub-complex, the ATP-binding activity of MCM6 is critical for helicase function: mutagenesis of its conserved Walker A/B ATPase motifs abolishes unwinding, and its ATPase active site (with MCM2) contributes to the Mcm2/5 'gate' that gates the hexameric ring, while ssDNA binding maps to MCM4 [#0, #8]. MCM6 is loaded onto chromatin at replication origins during G1 phase in an ORC-dependent manner, where it associates with other MCM subunits and exists in both chromatin-bound and soluble forms across the cell cycle [#2, #3, #4, #9]. Origin licensing is mediated by a direct interaction between Cdt1 and the C-terminal Cdt1-binding domain (CBD) of MCM6, a domain that adopts a winged-helix fold; small molecules that occupy the CBD competitively block the MCM6-CDT1 interaction, arresting cells in G0/G1 [#5, #11, #14, #21]. Helicase activity is restrained by CDK- and EBV-PK-mediated phosphorylation, and MCM6 additionally couples replication to genome surveillance by binding the checkpoint mediator Mrc1/Claspin to activate the MMS-induced replication checkpoint and by binding the BLM helicase in a cell-cycle-phase-specific manner to limit replication fork speed [#6, #7, #10]. MCM6 abundance is controlled transcriptionally by E2F and, in cancer, by YAP-TEAD and c-Myc, and post-translationally by ubiquitination (RNF125, RNF8) and SIRT7-mediated K599 crotonylation that accompanies MCM2-7 disassembly under replication stress [#1, #12, #13, #15, #19]. A de novo missense variant disrupting a zinc-binding cysteine of the MCM6 zinc finger impairs ciliogenesis and proliferation in patient fibroblasts, linking MCM6 dysfunction to human disease [#22].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that human MCM6 is a physical component of a heteromeric MCM complex and that its localization is cell-cycle regulated, peaking at G1/S, defining it as a replication-associated factor.\",\n      \"evidence\": \"Co-immunoprecipitation with MCM2/MCM7 and subcellular fractionation across synchronized cells\",\n      \"pmids\": [\"9286856\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve the stoichiometry or which subunits contact MCM6 directly\", \"No functional readout of the complex\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined the enzymatic core of MCM6 by showing its ATP-binding activity is essential for helicase function of the MCM4-6-7 sub-complex, separating helicase from ssDNA-binding activities.\",\n      \"evidence\": \"Recombinant mouse MCM4/6/7 with Walker A/B motif mutagenesis and in vitro helicase and ssDNA-binding assays\",\n      \"pmids\": [\"10567526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MCM4-6-7 sub-complex is not the physiological MCM2-7 ring\", \"Did not address how ATP hydrolysis is coupled to translocation\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Connected MCM6 expression to cell-cycle entry by showing E2F drives its transcription, explaining serum-responsive induction of licensing capacity.\",\n      \"evidence\": \"Promoter mutagenesis, luciferase reporters and forced E2F1 expression in REF52 cells\",\n      \"pmids\": [\"10327050\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish which endogenous E2F member dominates in vivo\", \"No link to protein-level licensing output\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Showed origin loading of MCM6 in vivo by mapping the fission yeast ortholog to specific replication origins in an ORC-dependent, G1/S-restricted manner.\",\n      \"evidence\": \"Cell fractionation, spread-nuclei immunostaining and ChIP at ars2004/ars3002 in S. pombe\",\n      \"pmids\": [\"10490657\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ortholog system; human origin engagement inferred\", \"Loading mechanism beyond ORC not resolved here\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified the direct licensing contact by mapping a Cdt1-MCM6 interaction through Cdt1's C-terminal region, distinguishing it from the geminin-regulated DNA-binding domain.\",\n      \"evidence\": \"Yeast two-hybrid, purified-protein binding and domain mapping of mouse Cdt1\",\n      \"pmids\": [\"12192004\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not localize the reciprocal MCM6 binding surface in this study\", \"Geminin's effect on loading kinetics not measured\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined phosphoregulation of the helicase, showing CDK and EBV-PK phosphorylation (including of MCM6) inactivates the MCM4-6-7 complex.\",\n      \"evidence\": \"In vitro kinase assays with CDK2/cyclin A and EBV-PK plus MCM4 mutagenesis and helicase readouts\",\n      \"pmids\": [\"17005684\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MCM6 phosphosites not precisely mapped\", \"Physiological relevance in the MCM2-7 ring not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed a checkpoint-sensing role by mapping a direct Mcm6 C-terminus to Mrc1 coiled-coil interaction required specifically for the MMS-induced replication checkpoint.\",\n      \"evidence\": \"Two-hybrid, reciprocal Co-IP, point mutagenesis and fusion-rescue genetics in budding yeast\",\n      \"pmids\": [\"19620285\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"HU-induced checkpoint is independent of this interaction\", \"Human Claspin equivalence not directly tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Refined the catalytic architecture by showing the Mcm6/2 ATPase active site contributes to the Mcm2/5 gate that modulates Mcm2-7 helicase activity.\",\n      \"evidence\": \"Walker B and arginine-finger mutagenesis across individual active sites with in vitro ATPase/helicase assays in S. cerevisiae\",\n      \"pmids\": [\"20484375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Gate opening dynamics not directly visualized\", \"Coupling to DNA loading versus unwinding not separated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linked chromatin-bound MCM6 levels to commitment past the restriction point, defining distinct G1 populations with different cycling fates.\",\n      \"evidence\": \"Flow cytometry with detergent extraction and serum-withdrawal/drug arrest kinetics across cell lines\",\n      \"pmids\": [\"20398392\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Correlative rather than causal link to restriction-point control\", \"Molecular trigger for the high-Mcm6* state unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided structural identity for the licensing module by showing the MCM6 CBD adopts a winged-helix fold.\",\n      \"evidence\": \"Triple-resonance NMR chemical-shift assignments of human MCM6 CBD (BMRB 16396)\",\n      \"pmids\": [\"20623209\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Assignments only; no full structure or Cdt1-bound complex\", \"No mutagenesis of the binding interface in this work\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered a fork-speed control mechanism via phase-specific BLM-Mcm6 interactions, with the S-phase contact restraining replication speed and protecting genome integrity.\",\n      \"evidence\": \"BLM complex composition, domain-specific binding to N-/C-terminal Mcm6, replication speed and DSB-repair assays with BLM helicase mutants\",\n      \"pmids\": [\"34370039\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Switch mechanism between N-terminal and CBD sites not resolved\", \"How accelerated forks cause DSBs mechanistically unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked MCM6 to human Mendelian disease by showing a de novo zinc-finger cysteine variant impairs ciliogenesis and proliferation.\",\n      \"evidence\": \"Trio exome/genome sequencing and patient-derived fibroblast functional assays\",\n      \"pmids\": [\"37198333\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Helicase-level consequence of the variant not directly measured\", \"Mechanistic basis of the ciliary phenotype unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined PTM-based regulation of complex stability by identifying SIRT7-mediated K599 crotonylation, coupled to RNF8 ubiquitination, that accompanies MCM2-7 disassembly under replication stress.\",\n      \"evidence\": \"Site mapping, Kcr western blotting, knockdown replication assays and RNF8 ubiquitination assays\",\n      \"pmids\": [\"39477811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal order of crotonylation versus ubiquitination not established\", \"Direct effect of K599cr on helicase activity not measured\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified RNF125 as an E3 ligase that ubiquitinates MCM6 and drives proliferation through it in hepatocellular carcinoma.\",\n      \"evidence\": \"Pull-down, Co-IP, ubiquitination assay and MCM6 rescue co-transfection\",\n      \"pmids\": [\"38298426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ubiquitination is degradative or signaling not resolved\", \"Lysine target sites not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed MCM6 in oncogenic transcriptional circuitry as a direct YAP-TEAD target that feeds PI3K/Akt signaling and supports the ATR/Chk1 damage response.\",\n      \"evidence\": \"RNA-seq, ChIP-PCR, luciferase reporters, organoid/xenograft knockdown and pathway western blots in gastric cancer\",\n      \"pmids\": [\"36185598\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the PI3K/Akt link is direct or replication-dependent unclear\", \"Mechanism connecting MCM6 loss to ATR/Chk1 failure not detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a c-Myc/MCM6 positive feedback loop supporting DNA damage repair and cisplatin resistance.\",\n      \"evidence\": \"Knockdown/overexpression, ChIP for c-Myc on MCM6 promoter, ubiquitination and DDR assays, xenograft chemosensitivity\",\n      \"pmids\": [\"39805445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical basis of MCM6-promoted c-Myc stabilization unclear\", \"Single tumor context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated druggability of the MCM6 CBD with multiple small molecules that competitively block CDT1 binding and arrest cells, with MCM6/CDT1 overexpression rescuing toxicity.\",\n      \"evidence\": \"Modified ELISA, DARTS, molecular docking and cell-cycle/apoptosis assays with avermectin B1a and emodepside\",\n      \"pmids\": [\"39579921\", \"41203057\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Residue-level binding from docking not all confirmed structurally\", \"Selectivity over other winged-helix domains untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse cancer-context signaling roles (ERK/EMT, E2F1, decidualization) mechanistically derive from the core licensing/helicase function of MCM6 remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical link between MCM6 helicase activity and ERK/EMT outputs\", \"Whether non-replication roles reflect off-complex MCM6 is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [2, 4, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [0, 5, 8]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 9, 17]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [7, 10]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [7, 12]}\n    ],\n    \"complexes\": [\"MCM2-7 helicase\", \"MCM4-MCM6-MCM7 sub-complex\"],\n    \"partners\": [\"MCM2\", \"MCM7\", \"MCM4\", \"CDT1\", \"MRC1\", \"BLM\", \"RNF8\", \"RNF125\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}