{"gene":"MCM10","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1997,"finding":"Mcm10 (DNA43) is a nuclear protein that physically interacts with several members of the MCM2-7 family; loss of Mcm10 causes a dramatic reduction in DNA replication initiation at chromosomal origins and causes replication forks to pause during elongation through these same loci.","method":"Genetic analysis, two-dimensional DNA gel analysis, physical interaction assays in S. cerevisiae","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (2D gel, physical interaction, genetic), replicated across subsequent studies","pmids":["9154825"],"is_preprint":false},{"year":1992,"finding":"DNA43 (MCM10) is an essential gene required for entry into or completion of S phase in S. cerevisiae, identified as encoding a 59.6 kDa protein required for DNA synthesis.","method":"Temperature-sensitive mutant screen, DNA synthesis monitoring in synchronous populations, complementation cloning","journal":"Yeast","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic complementation and cell-cycle phenotype analysis, foundational identification paper replicated by subsequent work","pmids":["1514326"],"is_preprint":false},{"year":2000,"finding":"Mcm10 interacts physically with Mcm7 (a subunit of MCM2-7); diminished Mcm10–Mcm7 interaction inhibits replication initiation. Mcm10 mediates association of the MCM2-7 complex with replication origins, and interaction with Mcm7 is required for proper replication initiation and prompt release of origin-bound factors.","method":"Physical interaction assays, genetic epistasis (double-mutant rescue of mcm10-1/mcm7-1), chromatin association assays in S. cerevisiae","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic rescue, physical interaction, multiple orthogonal methods, replicated","pmids":["10783164"],"is_preprint":false},{"year":2002,"finding":"Xenopus Mcm10 chromatin binding requires chromatin-bound Mcm2-7 and is independent of Cdk2 and Cdc7. In the absence of Mcm10, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked, placing Mcm10 function after pre-RC assembly and before origin unwinding.","method":"Xenopus egg extract depletion/add-back, chromatin binding assays, plasmid supercoiling assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — depletion/rescue in cell-free system with multiple functional readouts, replicated across organisms","pmids":["11864598"],"is_preprint":false},{"year":2000,"finding":"Human Mcm10 (HsMcm10) associates with nuclease-resistant nuclear structures throughout S phase and dissociates in G2 phase. It interacts with human Orc2, Mcm2, and Mcm6 proteins.","method":"Nuclease-resistance fractionation, co-immunoprecipitation in COS-1 cells, yeast two-hybrid","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (fractionation, co-IP, two-hybrid) but single lab","pmids":["11095689"],"is_preprint":false},{"year":2003,"finding":"Fission yeast Cdc23/Mcm10 functions after pre-RC formation: its inactivation does not affect Mcm2 chromatin association (pre-RC formation) but blocks Cdc45 chromatin binding, placing Mcm10 function between pre-RC formation and Cdc45 loading.","method":"Degron allele inactivation, chromatin binding assays by cytological approach in S. pombe","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — tight degron, specific chromatin binding readout, consistent with Xenopus data from independent lab","pmids":["12972571"],"is_preprint":false},{"year":2003,"finding":"Fission yeast Cdc23p (Mcm10) interacts with both the Mcm complex (via selective binding to Mcm467 subunits) and Dfp1p (the regulatory subunit of Dfp1-Hsk1/Dbf4-Cdc7 kinase). Cdc23p is required for efficient phosphorylation of Mcm2p and Mcm4p within the six-subunit Mcm complex by Dfp1-Hsk1 kinase in vitro; this activity requires the N-terminus of Cdc23p.","method":"In vitro kinase assay with purified components, co-immunoprecipitation, truncation analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted kinase assay with purified proteins, truncation mapping, single lab","pmids":["12604790"],"is_preprint":false},{"year":2004,"finding":"Mcm10 binding to replication origins in budding yeast is cell-cycle regulated and dependent on Mcm2-7. Mcm10 is required to maintain steady-state levels of the catalytic subunit of DNA polymerase-alpha (pol-alpha); depletion of Mcm10 during S phase causes degradation of pol-alpha without affecting Cdc45.","method":"ChIP, temperature-sensitive degron (mcm10-td) mutants, immunoblotting in S. cerevisiae","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional depletion with specific phenotypic readout, replicated across organisms and labs","pmids":["15494305"],"is_preprint":false},{"year":2004,"finding":"Mcm10 physically interacts with Cdc45 and facilitates recruitment of Cdc45 to the ARS1 origin. Overexpression of either Mcm10 or Cdc45 suppresses the growth defect of mcm10-1, and the physical Cdc45-Mcm10 interaction is disrupted in the mcm10-1 mutant.","method":"ChIP at ARS1, co-immunoprecipitation, overexpression suppression analysis in S. cerevisiae","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and ChIP with genetic suppression, single lab","pmids":["15201046"],"is_preprint":false},{"year":2006,"finding":"Mcm10 contains a PCNA-interacting (PIP) box and directly interacts with PCNA; only the diubiquitinated form of Mcm10 binds PCNA. Diubiquitination of Mcm10 is cell-cycle regulated (appears in late G1, persists through S phase) and associated with chromatin. A PIP-box mutation (Y245A) abolishing PCNA interaction is lethal, rescued by a compensatory PCNA mutation.","method":"PIP-box mutational analysis, in vitro pulldown, co-IP, cell-cycle fractionation, genetic rescue in S. cerevisiae","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with functional rescue, in vitro pulldown confirming direct interaction, multiple orthogonal methods","pmids":["16782870"],"is_preprint":false},{"year":2006,"finding":"A conserved Hsp10-like domain in Mcm10 is required to stabilize the catalytic subunit of DNA polymerase-alpha (Cdc17/Pol1); single residue substitution in this domain dramatically reduces Cdc17 steady-state levels. Mcm10 co-overexpression stabilizes Cdc17 subject to rapid degradation, consistent with Mcm10 acting as a nuclear chaperone for Cdc17.","method":"Temperature-sensitive degron mutants, overexpression rescue, site-directed mutagenesis, immunoblotting in S. cerevisiae","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis of conserved domain + rescue experiment, multiple alleles tested","pmids":["16675460"],"is_preprint":false},{"year":2007,"finding":"And-1/Ctf4 interacts with Mcm10 (which associates with MCM2-7) and with the p180 subunit of DNA pol alpha. In Xenopus egg extracts, And-1 chromatin loading requires Mcm10; antibody disrupting the Mcm10-And-1 interaction prevents loading of And-1 and pol alpha, inhibiting DNA synthesis. And-1 is essential for DNA synthesis and stability of p180.","method":"Co-immunoprecipitation, Xenopus egg extract depletion/antibody inhibition, chromatin binding assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, functional antibody disruption in Xenopus extracts, multiple organisms tested","pmids":["17761813"],"is_preprint":false},{"year":2007,"finding":"Human Mcm10 interacts with and stabilizes the catalytic subunit of pol-alpha (p180) in human HeLa cells; siRNA-mediated depletion of Mcm10 causes degradation of p180 with similar kinetics, while the regulatory p68 subunit is unaffected. Simultaneous loss of Mcm10 and p180 causes S phase entry inhibition and DNA damage.","method":"siRNA knockdown, immunoblotting, cell-cycle analysis in HeLa cells","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — siRNA depletion in human cells with specific molecular phenotype, consistent with yeast and Xenopus data","pmids":["17699597"],"is_preprint":false},{"year":2007,"finding":"Human MCM10 forms a ring-shaped hexameric structure with large central and smaller lateral channels and a system of inner chambers, as determined by electron microscopy.","method":"Electron microscopy and single-particle analysis of purified human MCM10","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — EM structure without mutagenesis validation; single lab, no functional follow-up in the same study","pmids":["17823614"],"is_preprint":false},{"year":2007,"finding":"The highly conserved internal domain (Mcm10-ID) from Xenopus binds ssDNA via an OB-fold followed by a variant zinc finger. NMR chemical shift perturbation and mutagenesis of DNA-binding residues confirm the ssDNA-binding surface; corresponding mutations in S. cerevisiae increase sensitivity to replication stress.","method":"X-ray crystallography of Mcm10-ID, NMR chemical shift perturbation, site-directed mutagenesis, replication stress assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure + NMR + mutagenesis with functional validation across two organisms","pmids":["19081065"],"is_preprint":false},{"year":2007,"finding":"Xenopus Mcm10 has three structural domains: N-terminal (homodimerization), internal (ID), and C-terminal (CTD). The ID and CTD both bind ssDNA and dsDNA with low micromolar affinity and independently bind the N-terminal 323 residues of the pol alpha p180 subunit. Structural integrity of ID and CTD depends on bound zinc.","method":"Limited proteolysis, analytical ultracentrifugation, DNA binding assays, atomic absorption spectroscopy, co-IP with pol alpha fragments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biochemical methods with purified domains, zinc validated independently","pmids":["18065420"],"is_preprint":false},{"year":2009,"finding":"Human RECQ4 forms a complex with MCM10, MCM2-7 helicase, CDC45, and GINS on chromatin in a cell-cycle-regulated manner. MCM10 is essential for the integrity of the RECQ4-MCM replicative helicase complex; MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity. This interaction may be modulated by CDK phosphorylation.","method":"Purification of chromatin-bound complexes, co-immunoprecipitation, in vitro DNA unwinding assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — purified complex from human cells, in vitro helicase assay, co-IP; single lab with multiple orthogonal methods","pmids":["19696745"],"is_preprint":false},{"year":2009,"finding":"CMG complex formation in human cells requires MCM10, RecQL4, and Ctf4/And-1 in addition to the CMG components. CMG assembly occurs only after G1/S transition and requires CDK and Cdc7 kinase activities; depletion of MCM10 by siRNA abolishes CMG complex formation.","method":"Bimolecular fluorescence complementation (BiFC) in HeLa cells, siRNA knockdown","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BiFC and siRNA in human cells with specific complex formation readout; single lab","pmids":["19805216"],"is_preprint":false},{"year":2009,"finding":"Mcm10 ID and CTD both bind the OB-fold cleft of Mcm10-ID for ssDNA; ssDNA and pol-alpha p180 compete for binding to the OB-fold of Mcm10-ID. The minimal Mcm10-binding site on p180 maps to residues 286-310 within the p180 N-terminal domain, suggesting a handoff mechanism for pol alpha loading.","method":"X-ray crystallography of Mcm10-ID·ssDNA complex, NMR chemical shift perturbation, fluorescence spectroscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure + NMR + fluorescence spectroscopy, competition experiment with purified components","pmids":["19608746"],"is_preprint":false},{"year":2009,"finding":"Saccharomyces cerevisiae Mcm10 binds both dsDNA and ssDNA stably; on longer dsDNA, multiple copies cooperate to assemble a large nucleoprotein complex with ~21-24 bp spacing. On ssDNA, approximately 3 copies of Mcm10 assemble per short ssDNA oligomer, suggesting multisubunit complex formation on ssDNA.","method":"EMSA, surface plasmon resonance with purified ScMcm10","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assay with purified protein, two orthogonal methods; single lab","pmids":["19605346"],"is_preprint":false},{"year":2001,"finding":"Human Mcm10 protein levels are regulated by cell-cycle-dependent proteolysis (proteasome-mediated degradation in late M/G1 phase) and by hyperphosphorylation in G2/M phase; Mcm10 binds chromatin at the onset of S phase and dissociates in G2/M.","method":"Cell synchronization, immunoblotting, proteasome inhibitor treatment, lambda phosphatase treatment, chromatin fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cell-cycle synchronization methods, phosphatase treatment confirming phosphorylation, proteasome inhibitor confirming proteolysis","pmids":["11602595"],"is_preprint":false},{"year":2004,"finding":"Human Mcm10 localizes to discrete nuclear foci during S phase; GFP-Mcm10 foci patterns (early S: throughout nucleus; mid S: nuclear periphery/nucleolus; late S: nucleoli) precede changes in replication foci by 30-60 min, suggesting Mcm10 is temporarily recruited to replication sites before they replicate and dissociates after pre-RC activation.","method":"GFP live-cell imaging in stable HeLa cell lines, pulse-labeling with BrdU to mark active replication foci","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with functional implication (temporal precedence over replication), single lab","pmids":["15136575"],"is_preprint":false},{"year":2010,"finding":"Solution NMR structure of the Xenopus Mcm10 C-terminal domain (CTD) reveals two zinc binding motifs; only the N-terminal CCCH-type zinc motif binds ssDNA, while the CCCC-type motif is structurally similar to the zinc ribbon in MCM helicase N-terminal oligomerization domain but does not bind DNA. The ID and CTD are structurally independent in solution.","method":"Solution NMR, NMR chemical shift perturbation, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with chemical shift perturbation and mutagenesis for functional validation","pmids":["20489205"],"is_preprint":false},{"year":2012,"finding":"Yeast Mcm10 interacts preferentially with the inactive Mcm2-7 double hexamer loaded at origins (not with the replisome). When Mcm10 is acutely degraded, CMG (Cdc45 and GINS) still assembles on Mcm2-7 at origins, but origin DNA unwinding is blocked — establishing a novel step after CMG assembly requiring Mcm10. Mcm10 also chaperones pol alpha in both yeast and human cells.","method":"Novel auxin-inducible degron (AID) for acute depletion, ChIP, biochemical fractionation in S. cerevisiae and human cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — acute conditional depletion with multiple ChIP readouts; orthogonal to two simultaneous independent studies from different labs","pmids":["22433841"],"is_preprint":false},{"year":2012,"finding":"In fission yeast, depletion of Mcm10 to <0.5% does not affect origin loading of Mcm2-7, Cdc45, or GINS, but impairs RPA and DNA polymerase recruitment, demonstrating that Mcm10 is required for origin DNA unwinding after CMG loading. A conserved zinc finger in Mcm10 is required for RPA loading.","method":"Promoter shut-off combined with auxin-inducible degron (off-AID), ChIP, zinc finger mutagenesis in S. pombe","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — extremely efficient depletion (<0.5%), zinc finger mutagenesis, multiple ChIP readouts; independent of yeast study above","pmids":["22433840"],"is_preprint":false},{"year":2012,"finding":"In budding yeast, depletion of Mcm10 using auxin-inducible degron assembles a stable CMG complex at origins but prevents CMG translocation, RPA loading, and intra-S checkpoint activation. Mcm10 associates with origins during initiation in an S-cyclin-dependent kinase- and Cdc45-dependent manner.","method":"Auxin-inducible degron, ChIP, two-dimensional gel electrophoresis in S. cerevisiae","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — acute conditional depletion with multiple independent molecular readouts; corroborated by two simultaneous independent studies","pmids":["22285032"],"is_preprint":false},{"year":2003,"finding":"Mcm10 self-assembles into large homocomplexes (~800 kDa) mediated by a conserved 210 aa domain containing a novel zinc finger (CX10CX11CX2H) essential for homocomplex formation; cysteine/histidine mutations that abolish zinc finger cause defects in homocomplex assembly, DNA replication, and cell growth.","method":"Co-immunoprecipitation, gel filtration, zinc finger mutagenesis, cell growth assays in S. cerevisiae","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with functional consequence, biochemical characterization of complex size; single lab","pmids":["12844493"],"is_preprint":false},{"year":2013,"finding":"Mcm10 self-association is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain; crystal structure at 2.4 Å reveals a three-helix bundle consistent with dimeric and trimeric assemblies. Mutations at the subunit interface disrupt in vitro dimerization and in vivo self-interaction.","method":"X-ray crystallography, analytical ultracentrifugation, yeast two-hybrid, site-directed mutagenesis","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure + AUC + mutagenesis with functional in vivo validation","pmids":["23894664"],"is_preprint":false},{"year":2013,"finding":"Human SIRT1 deacetylase binds and deacetylates Mcm10 both in vivo and in vitro, modulating Mcm10 stability and DNA binding ability. The two DNA-binding domains (ID and CTD) are regulated differently by acetylation/deacetylation; Mcm10 and SIRT1 act synergistically for replication fork initiation.","method":"Co-immunoprecipitation, in vitro deacetylation assay, DNA binding assays, domain-specific analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro deacetylation assay + co-IP, but functional interpretation relies on synergy without direct mutagenesis of acetylation sites","pmids":["23449222"],"is_preprint":false},{"year":2014,"finding":"RBBP6 ubiquitin ligase ubiquitinates and destabilizes the transcriptional repressor ZBTB38, which negatively regulates MCM10 levels on chromatin. Cells lacking RBBP6 accumulate ZBTB38, reduce MCM10 chromatin levels, experience reduced replication fork progression and increased damage at common fragile sites.","method":"siRNA knockdown, ubiquitination assays, replication fork progression analysis, CFS analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway dissection with multiple knockdowns, ubiquitination assay; single lab","pmids":["24726359"],"is_preprint":false},{"year":2015,"finding":"HIV-1 Vpr indirectly binds MCM10 in a VprBP-dependent manner and enhances ubiquitination and proteasomal degradation of MCM10 via the Cul4-DDB1[VprBP] E3 ubiquitin ligase. G2/M-defective Vpr mutants cannot deplete MCM10, linking MCM10 degradation to Vpr-induced G2/M arrest.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, Vpr mutant analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + ubiquitination assay + genetic mutant correlation; single lab","pmids":["26032416"],"is_preprint":false},{"year":2012,"finding":"CRL4-DDB1-VprBP ubiquitin ligase mediates UV-stress-induced proteolysis of Mcm10. Depletion of DDB1, Roc1, or Cul4 abrogates UV-triggered Mcm10 proteolysis. Purified Cul4-Roc1-DDB1 complex ubiquitinates Mcm10 in vitro; VprBP is the substrate recognition subunit targeting Mcm10.","method":"siRNA knockdown, in vitro ubiquitination assay with purified components, immunoblotting","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination reconstitution + siRNA validation; single lab with two orthogonal methods","pmids":["22570418"],"is_preprint":false},{"year":2015,"finding":"Mcm10 is recruited to replication initiation sites via direct binding to MCM through its C-terminus. The Mcm10 binding site on MCM includes Mcm2 and Mcm6 subunits and overlaps with the Cdt1-binding site. Mcm10 exhibits low-affinity recruitment in absence of CMG assembly and high-affinity recruitment when CMG assembles; Mcm10 unable to bind MCM directly cannot support DNA replication.","method":"Pulldown with purified proteins, biochemical fractionation, replication reconstitution assay in budding yeast","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro pulldown with purified MCM double hexamers and single hexamers, functional replication assay; single lab","pmids":["26719337"],"is_preprint":false},{"year":2015,"finding":"Mcm10 co-purifies exclusively with endogenous MCM2-7 double hexamers (DHs) on chromatin in G1 phase. Deletion of the Mcm10 C-terminus (main interaction domain with MCM) causes growth and S-phase defects; Mcm10-MCM fusion restores function. Mcm10 interaction-deficient mutants show delayed DH dissolution during S phase, suggesting Mcm10 promotes Mcm2-7 remodeling.","method":"Cellular fractionation, endogenous DH purification, chromatin immunoprecipitation, Mcm10-MCM fusion genetic rescue in S. cerevisiae","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — purification of endogenous DHs, genetic fusion rescue, multiple orthogonal approaches","pmids":["26686640"],"is_preprint":false},{"year":2015,"finding":"Mcm10 directly interacts with the Mcm2-7 complex and Cdc45, recruits Cdc45 to Mcm2-7 in vitro, stimulates Mcm2 phosphorylation by DDK in vivo and in vitro, and promotes timely recruitment of Cdc45 and GINS to Mcm2-7 during early S phase.","method":"In vitro pulldown with purified proteins, auxin-inducible degron, in vitro DDK phosphorylation assay, ChIP","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — purified protein interactions + in vitro kinase assay + in vivo ChIP; single lab","pmids":["26582917"],"is_preprint":false},{"year":2014,"finding":"The N-terminus of Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress (UV or HU). Truncation of the Mcm10 N-terminus causes UV sensitivity; this is not enhanced by MEC3 deletion, placing Mcm10 and Mec3 in the same pathway for resistance to replication stress.","method":"Co-immunoprecipitation, truncation analysis, genetic epistasis (double-mutant analysis), UV sensitivity assays in S. cerevisiae","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP + genetic epistasis; single lab","pmids":["24972833"],"is_preprint":false},{"year":2017,"finding":"Mcm10 binds a conserved motif between the OB-fold and A subdomain of Mcm2; mutations predicted to expose this motif restore growth to conditional-lethal MCM10 mutants. Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7, stimulates replication elongation in vivo and in vitro, and a lethal allele that stimulates initial unwinding but is defective in elongation and CMG binding was identified.","method":"In vitro replication assay, CMG binding assay with purified proteins, mutagenesis, AID-based depletion in S. cerevisiae","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro replication reconstitution + mutagenesis + in vivo elongation assay, identification of separation-of-function allele","pmids":["28270517"],"is_preprint":false},{"year":2017,"finding":"Mcm10 binds CMG and greatly stimulates CMG helicase activity in vitro. Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking (lagging) strand without displacing the blocks, indicating Mcm10 isomerizes the CMG-DNA complex from a duplex-encircling to a strand-exclusion mode.","method":"In vitro helicase/translocation assays with purified CMG and Mcm10, roadblock bypass assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro helicase assay with purified proteins, mechanistic bypass demonstration","pmids":["28869037"],"is_preprint":false},{"year":2018,"finding":"Mcm10 has potent strand-annealing activity both alone and in complex with CMG; CMG-Mcm10 unwinds and reanneals single strands in vitro. Mcm10 inhibits fork regression by the SMARCAL1 fork reversal enzyme. Cross-linking mass spectrometry shows Mcm10 contacts six CMG subunits, with the Mcm10 DNA-binding region on the N-face of CMG, placing it at the fork junction.","method":"In vitro strand annealing assay, fork regression assay with purified SMARCAL1, cross-linking mass spectrometry of CMG-Mcm10 complex","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple in vitro biochemical assays + structural cross-linking MS, purified components","pmids":["30598452"],"is_preprint":false},{"year":2016,"finding":"In Xenopus egg extracts, depletion of >99% of Mcm10 does not prevent the bulk of DNA replication but reduces fork elongation rate; absence of Mcm10 or its CDK phosphorylation results in instability of replisome proteins on DNA, particularly under replication stress. Mcm10 is a CDK substrate but does not require phosphorylation for chromatin association.","method":"Xenopus egg extract depletion (>99%), DNA fiber analysis, replisome stability assay, phospho-mutant analysis","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — very deep depletion in Xenopus extracts, multiple readouts; single lab, somewhat contradicts other Xenopus data","pmids":["27327991"],"is_preprint":false},{"year":2013,"finding":"Human Mcm10 and Cdc45 interact directly: the Mcm10 CTD binds Cdc45 by co-immunoprecipitation and surface plasmon resonance; the ID interacts with Cdc45 only in presence of DNA. Both ID and CTD bind bubble and fork DNA structures preferentially and enhance Cdc45 DNA-binding affinity.","method":"Co-immunoprecipitation from cell-free extracts, surface plasmon resonance, domain truncation analysis, DNA binding assays with human proteins","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro SPR + co-IP with purified domains; single lab","pmids":["23750504"],"is_preprint":false},{"year":2015,"finding":"RecQL4 is required for the origin binding of Mcm10 and Ctf4 in human cells; their association with replication origins requires both CDK and DDK activities and the presence of the pre-replicative complex. Physical interactions and origin association are targeted by DNA damage checkpoint pathways.","method":"ChIP, siRNA knockdown, cell-cycle synchronization, checkpoint inhibition in human cells","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP with multiple conditions, siRNA; single lab","pmids":["25602958"],"is_preprint":false},{"year":2017,"finding":"Human MCM10 interaction with Mcm2-7 requires the domain containing amino acids 530-655, which overlaps with the domain required for stable chromatin retention. The conserved domain (aa 200-482) is essential for DNA replication; Mcm10 depletion reduces replication initiation frequency and impairs RPA, pol alpha, and PCNA chromatin loading without affecting Cdc45 and pol epsilon loading.","method":"siRNA + complementation with truncation constructs, chromatin fractionation, DNA fiber analysis in human HeLa cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping by truncation + chromatin fractionation; single lab in human cells","pmids":["28646110"],"is_preprint":false},{"year":2021,"finding":"BRCA2 associates with MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation after DNA damage, while having no impact on stability of stalled replication forks.","method":"Co-immunoprecipitation, siRNA knockdown, ssDNA gap detection assays, fiber assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating association + functional assays; single lab","pmids":["34645815"],"is_preprint":false},{"year":2021,"finding":"MCM10 deficiency causes chronic replication stress, genomic instability, and telomere erosion. Loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with ssDNA. Terminally-arrested forks in MCM10-deficient cells require endonucleolytic processing by MUS81; MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening.","method":"Patient fibroblast analysis, CRISPR modeling, MCM10 knockdown in NK cell lines, iPSC-derived NK cells, telomere FISH, DNA fiber assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including patient cells, iPSC modeling, double-mutant epistasis, molecular telomere analysis","pmids":["33712616"],"is_preprint":false},{"year":2023,"finding":"During DNA replication termination under topological stress in vertebrates, RTEL1 and MCM10 are highly enriched on chromatin during fork convergence and cooperate to promote fork convergence; they do not impact topoisomerase activity but promote fork progression through replication barriers.","method":"Proteomics in Xenopus egg extracts, chromatin enrichment, functional depletion assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics-based discovery + functional depletion in Xenopus extracts; single lab","pmids":["36807139"],"is_preprint":false},{"year":2016,"finding":"An Mcm10 mutant (Mcm10-m2,3,4) defective in ssDNA binding in vitro also fails to stimulate DDK phosphorylation of Mcm2 in vitro. Expression in yeast causes severe growth and replication defects, reduced DDK-phosphorylated Mcm2 and GINS association with Mcm2-7 in vivo. Defects in origin melting persist in the mcm5-bob1 bypass background, indicating ssDNA binding by Mcm10 is independently required for origin melting.","method":"In vitro DNA binding and DDK phosphorylation assays, genetic analysis with mcm5-bob1, ChIP in S. cerevisiae","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro assays combined with genetic epistasis (bypass mutant) and ChIP; single lab","pmids":["27751725"],"is_preprint":false},{"year":2017,"finding":"An intact Mcm10 coiled-coil (NTD) interaction surface is required for stimulating DDK phosphorylation of Mcm2, binding long ssDNA, and recruiting pol alpha to Mcm2-7 in vitro. Mcm10-4A NTD mutant causes severe replication defects and diminished origin melting and GINS recruitment even in the mcm5-bob1 bypass background.","method":"In vitro phosphorylation and pol alpha recruitment assays, mcm5-bob1 genetic epistasis, ChIP, growth assays in S. cerevisiae","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution + genetic epistasis (bypass mutant) + ChIP; single lab with multiple orthogonal methods","pmids":["28510759"],"is_preprint":false},{"year":2019,"finding":"Suramin and several analogues directly inhibit Mcm10 DNA binding in vitro (competition fluorescence polarization, surface plasmon resonance) and reduce replication products in an in vitro replication assay. Binding is confirmed by SPR and selectivity for Mcm10 over human RPA demonstrated for some analogues.","method":"Fluorescence polarization HTS, surface plasmon resonance, in vitro replication assay","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assays with purified protein, multiple orthogonal binding methods; single lab","pmids":["31409229"],"is_preprint":false},{"year":2024,"finding":"Deficiency of Mcm10 dramatically elevates (GAA)n repeat instability in yeast; live-cell microscopy shows increased replication fork stalling at the repeat in mcm10-1 cells. Viability of strains with (GAA)100 repeat at an essential chromosomal location strongly depends on Mcm10 function. Rad9 checkpoint activation promotes viability but initiates repeat expansions via pol delta; when RPA is depleted, breakage of under-replicated repetitive DNA occurs in G2/M.","method":"Repeat instability assays, live-cell microscopy of replication fork stalling, genetic analysis, RPA depletion in S. cerevisiae","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging + genetic assays with specific readouts; single lab","pmids":["39627228"],"is_preprint":false},{"year":2016,"finding":"S. cerevisiae Mcm10 ubiquitination maps primarily to lysine 372; mutation of K372 to arginine ablates ubiquitination of overexpressed protein and causes hydroxyurea sensitivity in S-phase checkpoint-compromised cells, linking Mcm10 ubiquitination to suppression of replication stress.","method":"Mass spectrometry mapping, site-directed mutagenesis, hydroxyurea sensitivity assay in S. cerevisiae","journal":"Biochemistry and biophysics reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — MS-based site mapping + mutagenesis + functional sensitivity assay; single lab","pmids":["28497125"],"is_preprint":false},{"year":2021,"finding":"A germline variant rs2274110 in MCM10 exon 15 increases SUMOylation levels at K669 of MCM10 protein mediated by SUMO2/3 enzymes, leading to aberrant MCM10 overexpression and increased genomic instability (DNA over-replication).","method":"Exome-wide association study followed by functional SUMOylation assays, site-directed mutagenesis of K669, cell proliferation and genome instability assays","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SUMOylation site mutagenesis + functional consequence; single lab","pmids":["34185429"],"is_preprint":false},{"year":2025,"finding":"Using single-molecule imaging and ensemble biochemistry, Mcm10 and RecQL4 act in a concerted manner to activate the replicative CMG helicase: Mcm10 binds first to inactive helicases and recruits RecQL4, which synergizes with Mcm10 to promote helicase activation. Mcm10 is not incorporated into replisomes and dissociates from origins during replication initiation. In the absence of Mcm10, RecQL4 is recruited to origins via interaction with the Mcm7 subunit.","method":"Single-molecule imaging, ensemble biochemistry with purified proteins, genetic interaction analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — single-molecule + ensemble biochemistry; preprint, not yet peer-reviewed","pmids":["40799587"],"is_preprint":true},{"year":2024,"finding":"In an in vitro SV40 replisome reconstitution, Mcm10 increases processivity of the replisome by stabilizing stalled replisomes and increasing their chances of restarting synthesis without altering the rate.","method":"Single-molecule in vitro reconstitution of SV40 replisome with purified human Mcm10","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro single-molecule reconstitution; single lab, heterologous system (SV40)","pmids":["38967018"],"is_preprint":false},{"year":2003,"finding":"Drosophila Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2, as well as with Cdc45 and HP1. RNAi depletion of Mcm10 in KC cells results in loss of DNA content and aberrant chromosome condensation.","method":"Yeast two-hybrid, RNAi depletion in Drosophila KC cells, DNA content analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two-hybrid + RNAi with specific phenotypes; single lab","pmids":["12808023"],"is_preprint":false},{"year":2003,"finding":"Fission yeast Cdc23/Mcm10 is bound to chromatin throughout the cell cycle in growing cells and only displaced during quiescence; re-establishment of chromatin binding upon return to growth is independent of pre-RC formation.","method":"Chromatin fractionation assays throughout cell cycle in S. pombe","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chromatin fractionation with clear cell-cycle conditions; single lab","pmids":["12972571"],"is_preprint":false},{"year":2008,"finding":"Mcm10 C-terminal domain mediates interaction with Sir2; mutations in the C-terminal 108 aa of Mcm10 destroy two-hybrid interactions with Sir2 and Sir3. Mcm10 mediates the interaction between replication proteins (Mcm3, Mcm7) and the silencing factor Sir2 via its C-terminal domain, contributing to heterochromatic silencing independently of DNA replication.","method":"Yeast two-hybrid, silencing reporter assays, genetic analysis in S. cerevisiae","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two-hybrid mapping + functional silencing assays; single lab","pmids":["19064704"],"is_preprint":false},{"year":2019,"finding":"Fission yeast Cdc23/Mcm10 mutations in the pol-alpha-recruitment and putative primase homology domain abrogate ribonucleotide imprint formation at the mat1 locus, suggesting Mcm10 plays a direct role in installing the ribonucleotide imprint in cooperation with pol alpha and Swi1.","method":"Genetic analysis of mcm10/cdc23 domain mutants, alkaline-labile imprint assay in S. pombe","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutation analysis with specific molecular readout; single lab","pmids":["30759238"],"is_preprint":false},{"year":2003,"finding":"Two bipartite NLSs in Mcm10 mediate its constitutive nuclear localization; either NLS alone is sufficient to direct Mcm10 (and GFP fused to it) into the nucleus.","method":"NLS mutation analysis, GFP fusion localization in S. cerevisiae","journal":"Current genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional NLS mapping with GFP reporter and mutational analysis; single lab","pmids":["13680157"],"is_preprint":false}],"current_model":"MCM10 is a conserved, non-enzymatic scaffold protein essential for eukaryotic DNA replication that operates in multiple steps: it binds the inactive MCM2-7 double hexamer at replication origins (via its C-terminus interacting with Mcm2 and Mcm6), facilitates Cdc45 recruitment and CMG assembly, recruits And-1/Ctf4, and then drives an origin-unwinding step after CMG assembly by isomerizing the CMG–DNA complex from a duplex-encircling to a strand-exclusion mode; throughout elongation, Mcm10 binds CMG (at the N-face via six subunit contacts), stimulates CMG helicase activity and replisome processivity, stabilizes Cdc45–GINS association with Mcm2-7, acts as a nuclear chaperone for the catalytic subunit of DNA polymerase-alpha (p180) preventing its proteasomal degradation, interacts with diubiquitinated PCNA via a PIP box to facilitate Okazaki fragment synthesis, inhibits SMARCAL1-mediated fork regression via its strand-annealing activity, suppresses PRIMPOL-mediated repriming in partnership with BRCA2, interacts with RECQ4 to stimulate DDK-mediated MCM2-7 phosphorylation and helicase activation, and is itself regulated by cell-cycle-dependent ubiquitination, phosphorylation, acetylation (controlled by SIRT1), SUMOylation, and UV-induced proteasomal degradation via the CRL4-DDB1-VprBP E3 ligase."},"narrative":{"mechanistic_narrative":"MCM10 is a conserved, essential nuclear scaffold protein that acts at the transition from replication licensing to origin firing, functioning after pre-replicative complex assembly but before origin DNA unwinding [PMID:1514326, PMID:11864598, PMID:12972571]. It is recruited to origins through direct contacts between its C-terminus and the inactive MCM2-7 double hexamer (engaging Mcm2 and Mcm6), with low-affinity binding before and high-affinity binding after CMG assembly [PMID:26719337, PMID:26686640, PMID:11095689]. Once bound, MCM10 facilitates Cdc45 recruitment and CMG formation, stabilizes Cdc45-GINS association with MCM2-7, and stimulates DDK-mediated phosphorylation of Mcm2 [PMID:15201046, PMID:26582917, PMID:19805216, PMID:28270517]. Acute-depletion studies established that its defining role is to drive the origin-unwinding step after a stable CMG has assembled: MCM10 loss permits CMG assembly but blocks DNA melting, CMG translocation, and RPA loading [PMID:22433841, PMID:22433840, PMID:22285032]. Structurally, MCM10 is a self-associating protein whose internal (ID) and C-terminal (CTD) domains use OB-folds and zinc motifs to bind ssDNA, dsDNA, and the p180 catalytic subunit of DNA polymerase-alpha, supporting a handoff that loads pol-alpha at origins, and its ssDNA-binding activity is independently required for origin melting [PMID:19081065, PMID:18065420, PMID:19608746, PMID:27751725, PMID:12844493, PMID:23894664]. MCM10 acts as a chaperone that stabilizes pol-alpha (p180/Cdc17) against degradation across yeast, Xenopus, and human cells, and it bridges the replisome to And-1/Ctf4 and RECQ4 to complete helicase activation [PMID:16675460, PMID:17699597, PMID:17761813, PMID:19696745, PMID:40799587]. During elongation MCM10 binds the N-face of CMG via multiple subunit contacts, stimulates helicase activity and replisome processivity, isomerizes CMG from duplex-encircling to strand-exclusion mode, and through strand-annealing activity inhibits SMARCAL1-mediated fork regression [PMID:28869037, PMID:30598452, PMID:38967018, PMID:28270517]. It also engages PCNA via a PIP box (only the diubiquitinated form binds) and partners with BRCA2 to suppress PRIMPOL-mediated repriming, linking it to lagging-strand synthesis and replication-stress tolerance [PMID:16782870, PMID:34645815]. MCM10 levels and activity are tightly controlled by cell-cycle-dependent proteolysis, phosphorylation, SIRT1-mediated deacetylation, SUMOylation, and CRL4-DDB1-VprBP-mediated ubiquitination, including UV-induced and HIV-1 Vpr-driven degradation [PMID:11602595, PMID:23449222, PMID:34185429, PMID:22570418, PMID:26032416]. MCM10 deficiency causes chronic replication stress, genomic instability, and telomere erosion through accumulation of aberrant fork structures [PMID:33712616].","teleology":[{"year":1992,"claim":"Established MCM10 as an essential gene whose loss blocks S-phase progression, defining it as a core DNA replication factor before any molecular function was known.","evidence":"Temperature-sensitive mutant screen and complementation cloning in S. cerevisiae","pmids":["1514326"],"confidence":"High","gaps":["No molecular function assigned","No interaction partners identified"]},{"year":1997,"claim":"Connected MCM10 physically and functionally to the MCM2-7 family, showing it is required both for initiation at origins and for fork progression through them.","evidence":"2D DNA gel analysis, physical interaction and genetic assays in S. cerevisiae","pmids":["9154825"],"confidence":"High","gaps":["Specific MCM subunit contacts not mapped","Mechanism of fork pausing unknown"]},{"year":2000,"claim":"Localized MCM10 function to origin loading of MCM2-7 via direct Mcm7 interaction, and identified conserved human interactions with Orc2, Mcm2, and Mcm6.","evidence":"Genetic epistasis, physical interaction and chromatin assays in S. cerevisiae; co-IP and two-hybrid in human cells","pmids":["10783164","11095689"],"confidence":"High","gaps":["Whether MCM10 acts before or after pre-RC formation unresolved at this stage","Human interaction data from single lab"]},{"year":2003,"claim":"Placed MCM10 epistatically between pre-RC formation and Cdc45 loading and revealed a role in DDK-dependent MCM phosphorylation, refining its temporal position in initiation.","evidence":"Degron inactivation and chromatin assays in S. pombe; in vitro kinase assay with Dfp1-Hsk1 and truncation mapping; two-hybrid/RNAi in Drosophila","pmids":["12972571","12604790","12808023"],"confidence":"High","gaps":["Direct demonstration that MCM10 stimulates unwinding not yet available","Kinase-stimulation mechanism unresolved"]},{"year":2004,"claim":"Defined two distinct activities: facilitating Cdc45 recruitment to origins and stabilizing the pol-alpha catalytic subunit against degradation.","evidence":"ChIP, co-IP, overexpression suppression and conditional degron in S. cerevisiae","pmids":["15201046","15494305"],"confidence":"Medium","gaps":["Molecular basis of pol-alpha stabilization not yet defined","Cdc45 interaction surface unmapped"]},{"year":2006,"claim":"Assigned the pol-alpha-stabilizing activity to a conserved Hsp10-like chaperone domain and linked MCM10 to PCNA via a PIP box dependent on diubiquitination.","evidence":"Domain mutagenesis with rescue and PIP-box mutational analysis with genetic rescue in S. cerevisiae","pmids":["16675460","16782870"],"confidence":"High","gaps":["Identity of the ubiquitin ligase for MCM10 diubiquitination unknown","Functional consequence of PCNA binding for Okazaki processing not directly tested"]},{"year":2007,"claim":"Resolved MCM10 domain architecture and DNA-/pol-alpha-binding surfaces, and integrated MCM10 into the And-1/Ctf4 axis for pol-alpha chromatin loading.","evidence":"Crystallography, NMR and biochemistry of Xenopus Mcm10 domains; EM of human hexamer; co-IP and Xenopus extract depletion for And-1; siRNA in HeLa for human p180 stabilization","pmids":["19081065","18065420","17823614","17761813","17699597"],"confidence":"High","gaps":["Hexameric EM structure lacks mutagenesis validation","Stoichiometry on DNA in vivo unclear"]},{"year":2009,"claim":"Established the structural handoff logic (ssDNA and pol-alpha compete for the ID OB-fold) and embedded MCM10 in CMG/RECQ4 helicase complex assembly in human cells.","evidence":"Crystallography of ID-ssDNA complex with competition assays; purified complex co-IP and in vitro unwinding for RECQ4; BiFC and siRNA for CMG formation; EMSA/SPR for cooperative DNA binding","pmids":["19608746","19696745","19805216","19605346"],"confidence":"High","gaps":["Timing of pol-alpha handoff relative to unwinding unresolved","RECQ4-MCM10 functional hierarchy not yet ordered"]},{"year":2012,"claim":"Three independent acute-depletion studies converged on a discrete post-CMG-assembly function: MCM10 is required for origin DNA unwinding, CMG translocation, and RPA loading, not for CMG assembly itself.","evidence":"Auxin-inducible and off-AID degron systems with ChIP and 2D gels in S. cerevisiae and S. pombe","pmids":["22433841","22433840","22285032"],"confidence":"High","gaps":["Biochemical mechanism of the unwinding step not yet reconstituted","Role of the zinc finger in RPA loading mechanistically unexplained"]},{"year":2015,"claim":"Mapped the MCM10-MCM2-7 interface to the C-terminus contacting Mcm2/Mcm6 (overlapping the Cdt1 site), distinguished low- versus high-affinity recruitment modes, and tied MCM10 to DDK stimulation and Cdc45/GINS stabilization.","evidence":"Pulldowns with purified MCM double/single hexamers, replication reconstitution, endogenous DH purification with fusion rescue, and in vitro DDK assays in S. cerevisiae; ChIP and siRNA for RecQL4-dependent recruitment in human cells","pmids":["26719337","26686640","26582917","25602958"],"confidence":"High","gaps":["How MCM10 promotes double-hexamer remodeling structurally undefined","Order of RecQL4 and MCM10 origin loading still debated"]},{"year":2017,"claim":"Reconstitution demonstrated that MCM10 stimulates CMG helicase activity, supports elongation and isomerizes CMG to a strand-exclusion mode, with separation-of-function alleles uncoupling initial unwinding from elongation/CMG binding.","evidence":"In vitro replication and helicase/roadblock-bypass assays with purified CMG and Mcm10, mutagenesis and AID depletion in S. cerevisiae","pmids":["28270517","28869037"],"confidence":"High","gaps":["Structural basis of the duplex-to-strand-exclusion isomerization unresolved","Contribution to lagging-strand engagement in vivo not fully defined"]},{"year":2018,"claim":"Defined MCM10's strand-annealing activity and its physical footprint on six CMG subunits at the fork junction, linking it to suppression of SMARCAL1-driven fork regression.","evidence":"In vitro strand-annealing and fork-regression assays with purified SMARCAL1, plus cross-linking mass spectrometry of CMG-Mcm10","pmids":["30598452"],"confidence":"High","gaps":["High-resolution structure of CMG-Mcm10 lacking","In vivo relevance of fork-regression inhibition not directly tested"]},{"year":2021,"claim":"Connected MCM10 to genome- and telomere-protective functions, including BRCA2-dependent suppression of PRIMPOL repriming and prevention of telomere erosion under replication stress.","evidence":"Co-IP, siRNA and ssDNA-gap/fiber assays for BRCA2-PRIMPOL; patient fibroblasts, CRISPR/iPSC modeling, telomere FISH and MUS81 epistasis for telomere phenotypes","pmids":["34645815","33712616"],"confidence":"High","gaps":["Direct demonstration that MCM10 disease mutations drive telomere defects in patients limited","Mechanism linking strand-annealing to PRIMPOL suppression not resolved"]},{"year":2024,"claim":"Extended MCM10 function to replisome processivity and repeat-tract stability, showing it stabilizes stalled replisomes and prevents fork stalling and instability at structured repeats.","evidence":"Single-molecule SV40 replisome reconstitution with human Mcm10; repeat-instability assays and live-cell fork-stalling imaging in S. cerevisiae","pmids":["38967018","39627228"],"confidence":"Medium","gaps":["SV40 is a heterologous system","Repeat-instability mechanism dissected in single lab"]},{"year":null,"claim":"How MCM10's multiple in vitro activities (origin melting, CMG isomerization, strand annealing, pol-alpha handoff, RECQ4 cooperation) are temporally coordinated and structurally integrated at the firing origin remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of the MCM10-CMG-origin intermediate","Order of RECQ4 versus MCM10 loading not definitively settled","Mechanism coupling unwinding to CMG translocation undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[14,15,18,19,40,46]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[14,18]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[10,12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[11,18,32,34]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[16,34,36,37]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,4,58]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[20,21,33,55]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[21]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[3,23,32,36,37]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[20,25,34]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[35,43,44]}],"complexes":["CMG helicase","MCM2-7 double hexamer"],"partners":["MCM2","MCM6","CDC45","RECQ4","WDHD1","POLA1","PCNA","BRCA2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7L590","full_name":"Protein MCM10 homolog","aliases":[],"length_aa":875,"mass_kda":98.2,"function":"Acts as a replication initiation factor that brings together the MCM2-7 helicase and the DNA polymerase alpha/primase complex in order to initiate DNA replication. Additionally, plays a role in preventing DNA damage during replication. Key effector of the RBBP6 and ZBTB38-mediated regulation of DNA-replication and common fragile sites stability; acts as a direct target of transcriptional repression by ZBTB38 (PubMed:24726359)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q7L590/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MCM10","classification":"Common Essential","n_dependent_lines":667,"n_total_lines":1208,"dependency_fraction":0.5521523178807947},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000065328","cell_line_id":"CID001800","localizations":[{"compartment":"nucleolus_gc","grade":3},{"compartment":"nucleoplasm","grade":3},{"compartment":"cytoplasmic","grade":2}],"interactors":[{"gene":"CSNK2A2","stoichiometry":0.2},{"gene":"MAP4","stoichiometry":0.2},{"gene":"EMC8","stoichiometry":0.2},{"gene":"PRAF2","stoichiometry":0.2},{"gene":"HIST1H2BN;HIST1H2BM;HIST1H2BH;HIST2H2BF;HIST1H2BC;HIST1H2BD;HIST1H2BK;H2BFS","stoichiometry":0.2},{"gene":"MCM6","stoichiometry":0.2},{"gene":"HIST1H4A","stoichiometry":0.2},{"gene":"HIST2H2AA3;HIST2H2AC","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001800","total_profiled":1310},"omim":[{"mim_id":"619313","title":"IMMUNODEFICIENCY 80 WITH OR WITHOUT CONGENITAL CARDIOMYOPATHY; IMD80","url":"https://www.omim.org/entry/619313"},{"mim_id":"609357","title":"MINICHROMOSOME MAINTENANCE COMPLEX COMPONENT 10; MCM10","url":"https://www.omim.org/entry/609357"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"bone marrow","ntpm":8.7},{"tissue":"lymphoid tissue","ntpm":7.5}],"url":"https://www.proteinatlas.org/search/MCM10"},"hgnc":{"alias_symbol":["PRO2249","CNA43","DNA43"],"prev_symbol":[]},"alphafold":{"accession":"Q7L590","domains":[{"cath_id":"2.40.50.140","chopping":"256-386","consensus_level":"high","plddt":87.9721,"start":256,"end":386},{"cath_id":"-","chopping":"390-433","consensus_level":"medium","plddt":86.5543,"start":390,"end":433},{"cath_id":"-","chopping":"772-848","consensus_level":"medium","plddt":91.0026,"start":772,"end":848}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L590","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L590-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L590-F1-predicted_aligned_error_v6.png","plddt_mean":66.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MCM10","jax_strain_url":"https://www.jax.org/strain/search?query=MCM10"},"sequence":{"accession":"Q7L590","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7L590.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7L590/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L590"}},"corpus_meta":[{"pmid":"17761813","id":"PMC_17761813","title":"Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication.","date":"2007","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/17761813","citation_count":180,"is_preprint":false},{"pmid":"15494305","id":"PMC_15494305","title":"Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha.","date":"2004","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/15494305","citation_count":172,"is_preprint":false},{"pmid":"19805216","id":"PMC_19805216","title":"Assembly of the Cdc45-Mcm2-7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins.","date":"2009","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/19805216","citation_count":162,"is_preprint":false},{"pmid":"11864598","id":"PMC_11864598","title":"Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45.","date":"2002","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/11864598","citation_count":158,"is_preprint":false},{"pmid":"10783164","id":"PMC_10783164","title":"Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins.","date":"2000","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/10783164","citation_count":142,"is_preprint":false},{"pmid":"9154825","id":"PMC_9154825","title":"A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.","date":"1997","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9154825","citation_count":124,"is_preprint":false},{"pmid":"19696745","id":"PMC_19696745","title":"MCM10 mediates RECQ4 association with MCM2-7 helicase complex during DNA replication.","date":"2009","source":"The EMBO 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interaction assays in S. cerevisiae\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (2D gel, physical interaction, genetic), replicated across subsequent studies\",\n      \"pmids\": [\"9154825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"DNA43 (MCM10) is an essential gene required for entry into or completion of S phase in S. cerevisiae, identified as encoding a 59.6 kDa protein required for DNA synthesis.\",\n      \"method\": \"Temperature-sensitive mutant screen, DNA synthesis monitoring in synchronous populations, complementation cloning\",\n      \"journal\": \"Yeast\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic complementation and cell-cycle phenotype analysis, foundational identification paper replicated by subsequent work\",\n      \"pmids\": [\"1514326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Mcm10 interacts physically with Mcm7 (a subunit of MCM2-7); diminished Mcm10–Mcm7 interaction inhibits replication initiation. Mcm10 mediates association of the MCM2-7 complex with replication origins, and interaction with Mcm7 is required for proper replication initiation and prompt release of origin-bound factors.\",\n      \"method\": \"Physical interaction assays, genetic epistasis (double-mutant rescue of mcm10-1/mcm7-1), chromatin association assays in S. cerevisiae\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic rescue, physical interaction, multiple orthogonal methods, replicated\",\n      \"pmids\": [\"10783164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Xenopus Mcm10 chromatin binding requires chromatin-bound Mcm2-7 and is independent of Cdk2 and Cdc7. In the absence of Mcm10, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked, placing Mcm10 function after pre-RC assembly and before origin unwinding.\",\n      \"method\": \"Xenopus egg extract depletion/add-back, chromatin binding assays, plasmid supercoiling assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — depletion/rescue in cell-free system with multiple functional readouts, replicated across organisms\",\n      \"pmids\": [\"11864598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Human Mcm10 (HsMcm10) associates with nuclease-resistant nuclear structures throughout S phase and dissociates in G2 phase. It interacts with human Orc2, Mcm2, and Mcm6 proteins.\",\n      \"method\": \"Nuclease-resistance fractionation, co-immunoprecipitation in COS-1 cells, yeast two-hybrid\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (fractionation, co-IP, two-hybrid) but single lab\",\n      \"pmids\": [\"11095689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Fission yeast Cdc23/Mcm10 functions after pre-RC formation: its inactivation does not affect Mcm2 chromatin association (pre-RC formation) but blocks Cdc45 chromatin binding, placing Mcm10 function between pre-RC formation and Cdc45 loading.\",\n      \"method\": \"Degron allele inactivation, chromatin binding assays by cytological approach in S. pombe\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tight degron, specific chromatin binding readout, consistent with Xenopus data from independent lab\",\n      \"pmids\": [\"12972571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Fission yeast Cdc23p (Mcm10) interacts with both the Mcm complex (via selective binding to Mcm467 subunits) and Dfp1p (the regulatory subunit of Dfp1-Hsk1/Dbf4-Cdc7 kinase). Cdc23p is required for efficient phosphorylation of Mcm2p and Mcm4p within the six-subunit Mcm complex by Dfp1-Hsk1 kinase in vitro; this activity requires the N-terminus of Cdc23p.\",\n      \"method\": \"In vitro kinase assay with purified components, co-immunoprecipitation, truncation analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted kinase assay with purified proteins, truncation mapping, single lab\",\n      \"pmids\": [\"12604790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Mcm10 binding to replication origins in budding yeast is cell-cycle regulated and dependent on Mcm2-7. Mcm10 is required to maintain steady-state levels of the catalytic subunit of DNA polymerase-alpha (pol-alpha); depletion of Mcm10 during S phase causes degradation of pol-alpha without affecting Cdc45.\",\n      \"method\": \"ChIP, temperature-sensitive degron (mcm10-td) mutants, immunoblotting in S. cerevisiae\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional depletion with specific phenotypic readout, replicated across organisms and labs\",\n      \"pmids\": [\"15494305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Mcm10 physically interacts with Cdc45 and facilitates recruitment of Cdc45 to the ARS1 origin. Overexpression of either Mcm10 or Cdc45 suppresses the growth defect of mcm10-1, and the physical Cdc45-Mcm10 interaction is disrupted in the mcm10-1 mutant.\",\n      \"method\": \"ChIP at ARS1, co-immunoprecipitation, overexpression suppression analysis in S. cerevisiae\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and ChIP with genetic suppression, single lab\",\n      \"pmids\": [\"15201046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Mcm10 contains a PCNA-interacting (PIP) box and directly interacts with PCNA; only the diubiquitinated form of Mcm10 binds PCNA. Diubiquitination of Mcm10 is cell-cycle regulated (appears in late G1, persists through S phase) and associated with chromatin. A PIP-box mutation (Y245A) abolishing PCNA interaction is lethal, rescued by a compensatory PCNA mutation.\",\n      \"method\": \"PIP-box mutational analysis, in vitro pulldown, co-IP, cell-cycle fractionation, genetic rescue in S. cerevisiae\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with functional rescue, in vitro pulldown confirming direct interaction, multiple orthogonal methods\",\n      \"pmids\": [\"16782870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"A conserved Hsp10-like domain in Mcm10 is required to stabilize the catalytic subunit of DNA polymerase-alpha (Cdc17/Pol1); single residue substitution in this domain dramatically reduces Cdc17 steady-state levels. Mcm10 co-overexpression stabilizes Cdc17 subject to rapid degradation, consistent with Mcm10 acting as a nuclear chaperone for Cdc17.\",\n      \"method\": \"Temperature-sensitive degron mutants, overexpression rescue, site-directed mutagenesis, immunoblotting in S. cerevisiae\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis of conserved domain + rescue experiment, multiple alleles tested\",\n      \"pmids\": [\"16675460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"And-1/Ctf4 interacts with Mcm10 (which associates with MCM2-7) and with the p180 subunit of DNA pol alpha. In Xenopus egg extracts, And-1 chromatin loading requires Mcm10; antibody disrupting the Mcm10-And-1 interaction prevents loading of And-1 and pol alpha, inhibiting DNA synthesis. And-1 is essential for DNA synthesis and stability of p180.\",\n      \"method\": \"Co-immunoprecipitation, Xenopus egg extract depletion/antibody inhibition, chromatin binding assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, functional antibody disruption in Xenopus extracts, multiple organisms tested\",\n      \"pmids\": [\"17761813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human Mcm10 interacts with and stabilizes the catalytic subunit of pol-alpha (p180) in human HeLa cells; siRNA-mediated depletion of Mcm10 causes degradation of p180 with similar kinetics, while the regulatory p68 subunit is unaffected. Simultaneous loss of Mcm10 and p180 causes S phase entry inhibition and DNA damage.\",\n      \"method\": \"siRNA knockdown, immunoblotting, cell-cycle analysis in HeLa cells\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — siRNA depletion in human cells with specific molecular phenotype, consistent with yeast and Xenopus data\",\n      \"pmids\": [\"17699597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human MCM10 forms a ring-shaped hexameric structure with large central and smaller lateral channels and a system of inner chambers, as determined by electron microscopy.\",\n      \"method\": \"Electron microscopy and single-particle analysis of purified human MCM10\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — EM structure without mutagenesis validation; single lab, no functional follow-up in the same study\",\n      \"pmids\": [\"17823614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The highly conserved internal domain (Mcm10-ID) from Xenopus binds ssDNA via an OB-fold followed by a variant zinc finger. NMR chemical shift perturbation and mutagenesis of DNA-binding residues confirm the ssDNA-binding surface; corresponding mutations in S. cerevisiae increase sensitivity to replication stress.\",\n      \"method\": \"X-ray crystallography of Mcm10-ID, NMR chemical shift perturbation, site-directed mutagenesis, replication stress assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure + NMR + mutagenesis with functional validation across two organisms\",\n      \"pmids\": [\"19081065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Xenopus Mcm10 has three structural domains: N-terminal (homodimerization), internal (ID), and C-terminal (CTD). The ID and CTD both bind ssDNA and dsDNA with low micromolar affinity and independently bind the N-terminal 323 residues of the pol alpha p180 subunit. Structural integrity of ID and CTD depends on bound zinc.\",\n      \"method\": \"Limited proteolysis, analytical ultracentrifugation, DNA binding assays, atomic absorption spectroscopy, co-IP with pol alpha fragments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biochemical methods with purified domains, zinc validated independently\",\n      \"pmids\": [\"18065420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Human RECQ4 forms a complex with MCM10, MCM2-7 helicase, CDC45, and GINS on chromatin in a cell-cycle-regulated manner. MCM10 is essential for the integrity of the RECQ4-MCM replicative helicase complex; MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity. This interaction may be modulated by CDK phosphorylation.\",\n      \"method\": \"Purification of chromatin-bound complexes, co-immunoprecipitation, in vitro DNA unwinding assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — purified complex from human cells, in vitro helicase assay, co-IP; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19696745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CMG complex formation in human cells requires MCM10, RecQL4, and Ctf4/And-1 in addition to the CMG components. CMG assembly occurs only after G1/S transition and requires CDK and Cdc7 kinase activities; depletion of MCM10 by siRNA abolishes CMG complex formation.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC) in HeLa cells, siRNA knockdown\",\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 and siRNA in human cells with specific complex formation readout; single lab\",\n      \"pmids\": [\"19805216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mcm10 ID and CTD both bind the OB-fold cleft of Mcm10-ID for ssDNA; ssDNA and pol-alpha p180 compete for binding to the OB-fold of Mcm10-ID. The minimal Mcm10-binding site on p180 maps to residues 286-310 within the p180 N-terminal domain, suggesting a handoff mechanism for pol alpha loading.\",\n      \"method\": \"X-ray crystallography of Mcm10-ID·ssDNA complex, NMR chemical shift perturbation, fluorescence spectroscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure + NMR + fluorescence spectroscopy, competition experiment with purified components\",\n      \"pmids\": [\"19608746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Saccharomyces cerevisiae Mcm10 binds both dsDNA and ssDNA stably; on longer dsDNA, multiple copies cooperate to assemble a large nucleoprotein complex with ~21-24 bp spacing. On ssDNA, approximately 3 copies of Mcm10 assemble per short ssDNA oligomer, suggesting multisubunit complex formation on ssDNA.\",\n      \"method\": \"EMSA, surface plasmon resonance with purified ScMcm10\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assay with purified protein, two orthogonal methods; single lab\",\n      \"pmids\": [\"19605346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Human Mcm10 protein levels are regulated by cell-cycle-dependent proteolysis (proteasome-mediated degradation in late M/G1 phase) and by hyperphosphorylation in G2/M phase; Mcm10 binds chromatin at the onset of S phase and dissociates in G2/M.\",\n      \"method\": \"Cell synchronization, immunoblotting, proteasome inhibitor treatment, lambda phosphatase treatment, chromatin fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cell-cycle synchronization methods, phosphatase treatment confirming phosphorylation, proteasome inhibitor confirming proteolysis\",\n      \"pmids\": [\"11602595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Mcm10 localizes to discrete nuclear foci during S phase; GFP-Mcm10 foci patterns (early S: throughout nucleus; mid S: nuclear periphery/nucleolus; late S: nucleoli) precede changes in replication foci by 30-60 min, suggesting Mcm10 is temporarily recruited to replication sites before they replicate and dissociates after pre-RC activation.\",\n      \"method\": \"GFP live-cell imaging in stable HeLa cell lines, pulse-labeling with BrdU to mark active replication foci\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with functional implication (temporal precedence over replication), single lab\",\n      \"pmids\": [\"15136575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Solution NMR structure of the Xenopus Mcm10 C-terminal domain (CTD) reveals two zinc binding motifs; only the N-terminal CCCH-type zinc motif binds ssDNA, while the CCCC-type motif is structurally similar to the zinc ribbon in MCM helicase N-terminal oligomerization domain but does not bind DNA. The ID and CTD are structurally independent in solution.\",\n      \"method\": \"Solution NMR, NMR chemical shift perturbation, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with chemical shift perturbation and mutagenesis for functional validation\",\n      \"pmids\": [\"20489205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Yeast Mcm10 interacts preferentially with the inactive Mcm2-7 double hexamer loaded at origins (not with the replisome). When Mcm10 is acutely degraded, CMG (Cdc45 and GINS) still assembles on Mcm2-7 at origins, but origin DNA unwinding is blocked — establishing a novel step after CMG assembly requiring Mcm10. Mcm10 also chaperones pol alpha in both yeast and human cells.\",\n      \"method\": \"Novel auxin-inducible degron (AID) for acute depletion, ChIP, biochemical fractionation in S. cerevisiae and human cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — acute conditional depletion with multiple ChIP readouts; orthogonal to two simultaneous independent studies from different labs\",\n      \"pmids\": [\"22433841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In fission yeast, depletion of Mcm10 to <0.5% does not affect origin loading of Mcm2-7, Cdc45, or GINS, but impairs RPA and DNA polymerase recruitment, demonstrating that Mcm10 is required for origin DNA unwinding after CMG loading. A conserved zinc finger in Mcm10 is required for RPA loading.\",\n      \"method\": \"Promoter shut-off combined with auxin-inducible degron (off-AID), ChIP, zinc finger mutagenesis in S. pombe\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — extremely efficient depletion (<0.5%), zinc finger mutagenesis, multiple ChIP readouts; independent of yeast study above\",\n      \"pmids\": [\"22433840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In budding yeast, depletion of Mcm10 using auxin-inducible degron assembles a stable CMG complex at origins but prevents CMG translocation, RPA loading, and intra-S checkpoint activation. Mcm10 associates with origins during initiation in an S-cyclin-dependent kinase- and Cdc45-dependent manner.\",\n      \"method\": \"Auxin-inducible degron, ChIP, two-dimensional gel electrophoresis in S. cerevisiae\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — acute conditional depletion with multiple independent molecular readouts; corroborated by two simultaneous independent studies\",\n      \"pmids\": [\"22285032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Mcm10 self-assembles into large homocomplexes (~800 kDa) mediated by a conserved 210 aa domain containing a novel zinc finger (CX10CX11CX2H) essential for homocomplex formation; cysteine/histidine mutations that abolish zinc finger cause defects in homocomplex assembly, DNA replication, and cell growth.\",\n      \"method\": \"Co-immunoprecipitation, gel filtration, zinc finger mutagenesis, cell growth assays in S. cerevisiae\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with functional consequence, biochemical characterization of complex size; single lab\",\n      \"pmids\": [\"12844493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mcm10 self-association is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain; crystal structure at 2.4 Å reveals a three-helix bundle consistent with dimeric and trimeric assemblies. Mutations at the subunit interface disrupt in vitro dimerization and in vivo self-interaction.\",\n      \"method\": \"X-ray crystallography, analytical ultracentrifugation, yeast two-hybrid, site-directed mutagenesis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure + AUC + mutagenesis with functional in vivo validation\",\n      \"pmids\": [\"23894664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human SIRT1 deacetylase binds and deacetylates Mcm10 both in vivo and in vitro, modulating Mcm10 stability and DNA binding ability. The two DNA-binding domains (ID and CTD) are regulated differently by acetylation/deacetylation; Mcm10 and SIRT1 act synergistically for replication fork initiation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deacetylation assay, DNA binding assays, domain-specific analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro deacetylation assay + co-IP, but functional interpretation relies on synergy without direct mutagenesis of acetylation sites\",\n      \"pmids\": [\"23449222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RBBP6 ubiquitin ligase ubiquitinates and destabilizes the transcriptional repressor ZBTB38, which negatively regulates MCM10 levels on chromatin. Cells lacking RBBP6 accumulate ZBTB38, reduce MCM10 chromatin levels, experience reduced replication fork progression and increased damage at common fragile sites.\",\n      \"method\": \"siRNA knockdown, ubiquitination assays, replication fork progression analysis, CFS analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway dissection with multiple knockdowns, ubiquitination assay; single lab\",\n      \"pmids\": [\"24726359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HIV-1 Vpr indirectly binds MCM10 in a VprBP-dependent manner and enhances ubiquitination and proteasomal degradation of MCM10 via the Cul4-DDB1[VprBP] E3 ubiquitin ligase. G2/M-defective Vpr mutants cannot deplete MCM10, linking MCM10 degradation to Vpr-induced G2/M arrest.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, Vpr mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + ubiquitination assay + genetic mutant correlation; single lab\",\n      \"pmids\": [\"26032416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CRL4-DDB1-VprBP ubiquitin ligase mediates UV-stress-induced proteolysis of Mcm10. Depletion of DDB1, Roc1, or Cul4 abrogates UV-triggered Mcm10 proteolysis. Purified Cul4-Roc1-DDB1 complex ubiquitinates Mcm10 in vitro; VprBP is the substrate recognition subunit targeting Mcm10.\",\n      \"method\": \"siRNA knockdown, in vitro ubiquitination assay with purified components, immunoblotting\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination reconstitution + siRNA validation; single lab with two orthogonal methods\",\n      \"pmids\": [\"22570418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Mcm10 is recruited to replication initiation sites via direct binding to MCM through its C-terminus. The Mcm10 binding site on MCM includes Mcm2 and Mcm6 subunits and overlaps with the Cdt1-binding site. Mcm10 exhibits low-affinity recruitment in absence of CMG assembly and high-affinity recruitment when CMG assembles; Mcm10 unable to bind MCM directly cannot support DNA replication.\",\n      \"method\": \"Pulldown with purified proteins, biochemical fractionation, replication reconstitution assay in budding yeast\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro pulldown with purified MCM double hexamers and single hexamers, functional replication assay; single lab\",\n      \"pmids\": [\"26719337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Mcm10 co-purifies exclusively with endogenous MCM2-7 double hexamers (DHs) on chromatin in G1 phase. Deletion of the Mcm10 C-terminus (main interaction domain with MCM) causes growth and S-phase defects; Mcm10-MCM fusion restores function. Mcm10 interaction-deficient mutants show delayed DH dissolution during S phase, suggesting Mcm10 promotes Mcm2-7 remodeling.\",\n      \"method\": \"Cellular fractionation, endogenous DH purification, chromatin immunoprecipitation, Mcm10-MCM fusion genetic rescue in S. cerevisiae\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — purification of endogenous DHs, genetic fusion rescue, multiple orthogonal approaches\",\n      \"pmids\": [\"26686640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Mcm10 directly interacts with the Mcm2-7 complex and Cdc45, recruits Cdc45 to Mcm2-7 in vitro, stimulates Mcm2 phosphorylation by DDK in vivo and in vitro, and promotes timely recruitment of Cdc45 and GINS to Mcm2-7 during early S phase.\",\n      \"method\": \"In vitro pulldown with purified proteins, auxin-inducible degron, in vitro DDK phosphorylation assay, ChIP\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — purified protein interactions + in vitro kinase assay + in vivo ChIP; single lab\",\n      \"pmids\": [\"26582917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The N-terminus of Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress (UV or HU). Truncation of the Mcm10 N-terminus causes UV sensitivity; this is not enhanced by MEC3 deletion, placing Mcm10 and Mec3 in the same pathway for resistance to replication stress.\",\n      \"method\": \"Co-immunoprecipitation, truncation analysis, genetic epistasis (double-mutant analysis), UV sensitivity assays in S. cerevisiae\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP + genetic epistasis; single lab\",\n      \"pmids\": [\"24972833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Mcm10 binds a conserved motif between the OB-fold and A subdomain of Mcm2; mutations predicted to expose this motif restore growth to conditional-lethal MCM10 mutants. Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7, stimulates replication elongation in vivo and in vitro, and a lethal allele that stimulates initial unwinding but is defective in elongation and CMG binding was identified.\",\n      \"method\": \"In vitro replication assay, CMG binding assay with purified proteins, mutagenesis, AID-based depletion in S. cerevisiae\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro replication reconstitution + mutagenesis + in vivo elongation assay, identification of separation-of-function allele\",\n      \"pmids\": [\"28270517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Mcm10 binds CMG and greatly stimulates CMG helicase activity in vitro. Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking (lagging) strand without displacing the blocks, indicating Mcm10 isomerizes the CMG-DNA complex from a duplex-encircling to a strand-exclusion mode.\",\n      \"method\": \"In vitro helicase/translocation assays with purified CMG and Mcm10, roadblock bypass assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro helicase assay with purified proteins, mechanistic bypass demonstration\",\n      \"pmids\": [\"28869037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Mcm10 has potent strand-annealing activity both alone and in complex with CMG; CMG-Mcm10 unwinds and reanneals single strands in vitro. Mcm10 inhibits fork regression by the SMARCAL1 fork reversal enzyme. Cross-linking mass spectrometry shows Mcm10 contacts six CMG subunits, with the Mcm10 DNA-binding region on the N-face of CMG, placing it at the fork junction.\",\n      \"method\": \"In vitro strand annealing assay, fork regression assay with purified SMARCAL1, cross-linking mass spectrometry of CMG-Mcm10 complex\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple in vitro biochemical assays + structural cross-linking MS, purified components\",\n      \"pmids\": [\"30598452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In Xenopus egg extracts, depletion of >99% of Mcm10 does not prevent the bulk of DNA replication but reduces fork elongation rate; absence of Mcm10 or its CDK phosphorylation results in instability of replisome proteins on DNA, particularly under replication stress. Mcm10 is a CDK substrate but does not require phosphorylation for chromatin association.\",\n      \"method\": \"Xenopus egg extract depletion (>99%), DNA fiber analysis, replisome stability assay, phospho-mutant analysis\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — very deep depletion in Xenopus extracts, multiple readouts; single lab, somewhat contradicts other Xenopus data\",\n      \"pmids\": [\"27327991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human Mcm10 and Cdc45 interact directly: the Mcm10 CTD binds Cdc45 by co-immunoprecipitation and surface plasmon resonance; the ID interacts with Cdc45 only in presence of DNA. Both ID and CTD bind bubble and fork DNA structures preferentially and enhance Cdc45 DNA-binding affinity.\",\n      \"method\": \"Co-immunoprecipitation from cell-free extracts, surface plasmon resonance, domain truncation analysis, DNA binding assays with human proteins\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro SPR + co-IP with purified domains; single lab\",\n      \"pmids\": [\"23750504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RecQL4 is required for the origin binding of Mcm10 and Ctf4 in human cells; their association with replication origins requires both CDK and DDK activities and the presence of the pre-replicative complex. Physical interactions and origin association are targeted by DNA damage checkpoint pathways.\",\n      \"method\": \"ChIP, siRNA knockdown, cell-cycle synchronization, checkpoint inhibition in human cells\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP with multiple conditions, siRNA; single lab\",\n      \"pmids\": [\"25602958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human MCM10 interaction with Mcm2-7 requires the domain containing amino acids 530-655, which overlaps with the domain required for stable chromatin retention. The conserved domain (aa 200-482) is essential for DNA replication; Mcm10 depletion reduces replication initiation frequency and impairs RPA, pol alpha, and PCNA chromatin loading without affecting Cdc45 and pol epsilon loading.\",\n      \"method\": \"siRNA + complementation with truncation constructs, chromatin fractionation, DNA fiber analysis in human HeLa cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping by truncation + chromatin fractionation; single lab in human cells\",\n      \"pmids\": [\"28646110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BRCA2 associates with MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation after DNA damage, while having no impact on stability of stalled replication forks.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, ssDNA gap detection assays, fiber assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating association + functional assays; single lab\",\n      \"pmids\": [\"34645815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MCM10 deficiency causes chronic replication stress, genomic instability, and telomere erosion. Loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with ssDNA. Terminally-arrested forks in MCM10-deficient cells require endonucleolytic processing by MUS81; MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening.\",\n      \"method\": \"Patient fibroblast analysis, CRISPR modeling, MCM10 knockdown in NK cell lines, iPSC-derived NK cells, telomere FISH, DNA fiber assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including patient cells, iPSC modeling, double-mutant epistasis, molecular telomere analysis\",\n      \"pmids\": [\"33712616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"During DNA replication termination under topological stress in vertebrates, RTEL1 and MCM10 are highly enriched on chromatin during fork convergence and cooperate to promote fork convergence; they do not impact topoisomerase activity but promote fork progression through replication barriers.\",\n      \"method\": \"Proteomics in Xenopus egg extracts, chromatin enrichment, functional depletion assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics-based discovery + functional depletion in Xenopus extracts; single lab\",\n      \"pmids\": [\"36807139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"An Mcm10 mutant (Mcm10-m2,3,4) defective in ssDNA binding in vitro also fails to stimulate DDK phosphorylation of Mcm2 in vitro. Expression in yeast causes severe growth and replication defects, reduced DDK-phosphorylated Mcm2 and GINS association with Mcm2-7 in vivo. Defects in origin melting persist in the mcm5-bob1 bypass background, indicating ssDNA binding by Mcm10 is independently required for origin melting.\",\n      \"method\": \"In vitro DNA binding and DDK phosphorylation assays, genetic analysis with mcm5-bob1, ChIP in S. cerevisiae\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro assays combined with genetic epistasis (bypass mutant) and ChIP; single lab\",\n      \"pmids\": [\"27751725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"An intact Mcm10 coiled-coil (NTD) interaction surface is required for stimulating DDK phosphorylation of Mcm2, binding long ssDNA, and recruiting pol alpha to Mcm2-7 in vitro. Mcm10-4A NTD mutant causes severe replication defects and diminished origin melting and GINS recruitment even in the mcm5-bob1 bypass background.\",\n      \"method\": \"In vitro phosphorylation and pol alpha recruitment assays, mcm5-bob1 genetic epistasis, ChIP, growth assays in S. cerevisiae\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution + genetic epistasis (bypass mutant) + ChIP; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28510759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Suramin and several analogues directly inhibit Mcm10 DNA binding in vitro (competition fluorescence polarization, surface plasmon resonance) and reduce replication products in an in vitro replication assay. Binding is confirmed by SPR and selectivity for Mcm10 over human RPA demonstrated for some analogues.\",\n      \"method\": \"Fluorescence polarization HTS, surface plasmon resonance, in vitro replication assay\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assays with purified protein, multiple orthogonal binding methods; single lab\",\n      \"pmids\": [\"31409229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Deficiency of Mcm10 dramatically elevates (GAA)n repeat instability in yeast; live-cell microscopy shows increased replication fork stalling at the repeat in mcm10-1 cells. Viability of strains with (GAA)100 repeat at an essential chromosomal location strongly depends on Mcm10 function. Rad9 checkpoint activation promotes viability but initiates repeat expansions via pol delta; when RPA is depleted, breakage of under-replicated repetitive DNA occurs in G2/M.\",\n      \"method\": \"Repeat instability assays, live-cell microscopy of replication fork stalling, genetic analysis, RPA depletion in S. cerevisiae\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging + genetic assays with specific readouts; single lab\",\n      \"pmids\": [\"39627228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"S. cerevisiae Mcm10 ubiquitination maps primarily to lysine 372; mutation of K372 to arginine ablates ubiquitination of overexpressed protein and causes hydroxyurea sensitivity in S-phase checkpoint-compromised cells, linking Mcm10 ubiquitination to suppression of replication stress.\",\n      \"method\": \"Mass spectrometry mapping, site-directed mutagenesis, hydroxyurea sensitivity assay in S. cerevisiae\",\n      \"journal\": \"Biochemistry and biophysics reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — MS-based site mapping + mutagenesis + functional sensitivity assay; single lab\",\n      \"pmids\": [\"28497125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A germline variant rs2274110 in MCM10 exon 15 increases SUMOylation levels at K669 of MCM10 protein mediated by SUMO2/3 enzymes, leading to aberrant MCM10 overexpression and increased genomic instability (DNA over-replication).\",\n      \"method\": \"Exome-wide association study followed by functional SUMOylation assays, site-directed mutagenesis of K669, cell proliferation and genome instability assays\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SUMOylation site mutagenesis + functional consequence; single lab\",\n      \"pmids\": [\"34185429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Using single-molecule imaging and ensemble biochemistry, Mcm10 and RecQL4 act in a concerted manner to activate the replicative CMG helicase: Mcm10 binds first to inactive helicases and recruits RecQL4, which synergizes with Mcm10 to promote helicase activation. Mcm10 is not incorporated into replisomes and dissociates from origins during replication initiation. In the absence of Mcm10, RecQL4 is recruited to origins via interaction with the Mcm7 subunit.\",\n      \"method\": \"Single-molecule imaging, ensemble biochemistry with purified proteins, genetic interaction analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule + ensemble biochemistry; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"40799587\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In an in vitro SV40 replisome reconstitution, Mcm10 increases processivity of the replisome by stabilizing stalled replisomes and increasing their chances of restarting synthesis without altering the rate.\",\n      \"method\": \"Single-molecule in vitro reconstitution of SV40 replisome with purified human Mcm10\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro single-molecule reconstitution; single lab, heterologous system (SV40)\",\n      \"pmids\": [\"38967018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Drosophila Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2, as well as with Cdc45 and HP1. RNAi depletion of Mcm10 in KC cells results in loss of DNA content and aberrant chromosome condensation.\",\n      \"method\": \"Yeast two-hybrid, RNAi depletion in Drosophila KC cells, DNA content analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid + RNAi with specific phenotypes; single lab\",\n      \"pmids\": [\"12808023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Fission yeast Cdc23/Mcm10 is bound to chromatin throughout the cell cycle in growing cells and only displaced during quiescence; re-establishment of chromatin binding upon return to growth is independent of pre-RC formation.\",\n      \"method\": \"Chromatin fractionation assays throughout cell cycle in S. pombe\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chromatin fractionation with clear cell-cycle conditions; single lab\",\n      \"pmids\": [\"12972571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Mcm10 C-terminal domain mediates interaction with Sir2; mutations in the C-terminal 108 aa of Mcm10 destroy two-hybrid interactions with Sir2 and Sir3. Mcm10 mediates the interaction between replication proteins (Mcm3, Mcm7) and the silencing factor Sir2 via its C-terminal domain, contributing to heterochromatic silencing independently of DNA replication.\",\n      \"method\": \"Yeast two-hybrid, silencing reporter assays, genetic analysis in S. cerevisiae\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid mapping + functional silencing assays; single lab\",\n      \"pmids\": [\"19064704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Fission yeast Cdc23/Mcm10 mutations in the pol-alpha-recruitment and putative primase homology domain abrogate ribonucleotide imprint formation at the mat1 locus, suggesting Mcm10 plays a direct role in installing the ribonucleotide imprint in cooperation with pol alpha and Swi1.\",\n      \"method\": \"Genetic analysis of mcm10/cdc23 domain mutants, alkaline-labile imprint assay in S. pombe\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutation analysis with specific molecular readout; single lab\",\n      \"pmids\": [\"30759238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Two bipartite NLSs in Mcm10 mediate its constitutive nuclear localization; either NLS alone is sufficient to direct Mcm10 (and GFP fused to it) into the nucleus.\",\n      \"method\": \"NLS mutation analysis, GFP fusion localization in S. cerevisiae\",\n      \"journal\": \"Current genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional NLS mapping with GFP reporter and mutational analysis; single lab\",\n      \"pmids\": [\"13680157\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MCM10 is a conserved, non-enzymatic scaffold protein essential for eukaryotic DNA replication that operates in multiple steps: it binds the inactive MCM2-7 double hexamer at replication origins (via its C-terminus interacting with Mcm2 and Mcm6), facilitates Cdc45 recruitment and CMG assembly, recruits And-1/Ctf4, and then drives an origin-unwinding step after CMG assembly by isomerizing the CMG–DNA complex from a duplex-encircling to a strand-exclusion mode; throughout elongation, Mcm10 binds CMG (at the N-face via six subunit contacts), stimulates CMG helicase activity and replisome processivity, stabilizes Cdc45–GINS association with Mcm2-7, acts as a nuclear chaperone for the catalytic subunit of DNA polymerase-alpha (p180) preventing its proteasomal degradation, interacts with diubiquitinated PCNA via a PIP box to facilitate Okazaki fragment synthesis, inhibits SMARCAL1-mediated fork regression via its strand-annealing activity, suppresses PRIMPOL-mediated repriming in partnership with BRCA2, interacts with RECQ4 to stimulate DDK-mediated MCM2-7 phosphorylation and helicase activation, and is itself regulated by cell-cycle-dependent ubiquitination, phosphorylation, acetylation (controlled by SIRT1), SUMOylation, and UV-induced proteasomal degradation via the CRL4-DDB1-VprBP E3 ligase.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MCM10 is a conserved, essential nuclear scaffold protein that acts at the transition from replication licensing to origin firing, functioning after pre-replicative complex assembly but before origin DNA unwinding [#1, #3, #5]. It is recruited to origins through direct contacts between its C-terminus and the inactive MCM2-7 double hexamer (engaging Mcm2 and Mcm6), with low-affinity binding before and high-affinity binding after CMG assembly [#32, #33, #4]. Once bound, MCM10 facilitates Cdc45 recruitment and CMG formation, stabilizes Cdc45-GINS association with MCM2-7, and stimulates DDK-mediated phosphorylation of Mcm2 [#8, #34, #17, #36]. Acute-depletion studies established that its defining role is to drive the origin-unwinding step after a stable CMG has assembled: MCM10 loss permits CMG assembly but blocks DNA melting, CMG translocation, and RPA loading [#23, #24, #25]. Structurally, MCM10 is a self-associating protein whose internal (ID) and C-terminal (CTD) domains use OB-folds and zinc motifs to bind ssDNA, dsDNA, and the p180 catalytic subunit of DNA polymerase-alpha, supporting a handoff that loads pol-alpha at origins, and its ssDNA-binding activity is independently required for origin melting [#14, #15, #18, #46, #26, #27]. MCM10 acts as a chaperone that stabilizes pol-alpha (p180/Cdc17) against degradation across yeast, Xenopus, and human cells, and it bridges the replisome to And-1/Ctf4 and RECQ4 to complete helicase activation [#10, #12, #11, #16, #52]. During elongation MCM10 binds the N-face of CMG via multiple subunit contacts, stimulates helicase activity and replisome processivity, isomerizes CMG from duplex-encircling to strand-exclusion mode, and through strand-annealing activity inhibits SMARCAL1-mediated fork regression [#37, #38, #53, #36]. It also engages PCNA via a PIP box (only the diubiquitinated form binds) and partners with BRCA2 to suppress PRIMPOL-mediated repriming, linking it to lagging-strand synthesis and replication-stress tolerance [#9, #43]. MCM10 levels and activity are tightly controlled by cell-cycle-dependent proteolysis, phosphorylation, SIRT1-mediated deacetylation, SUMOylation, and CRL4-DDB1-VprBP-mediated ubiquitination, including UV-induced and HIV-1 Vpr-driven degradation [#20, #28, #51, #31, #30]. MCM10 deficiency causes chronic replication stress, genomic instability, and telomere erosion through accumulation of aberrant fork structures [#44].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established MCM10 as an essential gene whose loss blocks S-phase progression, defining it as a core DNA replication factor before any molecular function was known.\",\n      \"evidence\": \"Temperature-sensitive mutant screen and complementation cloning in S. cerevisiae\",\n      \"pmids\": [\"1514326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No molecular function assigned\", \"No interaction partners identified\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Connected MCM10 physically and functionally to the MCM2-7 family, showing it is required both for initiation at origins and for fork progression through them.\",\n      \"evidence\": \"2D DNA gel analysis, physical interaction and genetic assays in S. cerevisiae\",\n      \"pmids\": [\"9154825\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific MCM subunit contacts not mapped\", \"Mechanism of fork pausing unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Localized MCM10 function to origin loading of MCM2-7 via direct Mcm7 interaction, and identified conserved human interactions with Orc2, Mcm2, and Mcm6.\",\n      \"evidence\": \"Genetic epistasis, physical interaction and chromatin assays in S. cerevisiae; co-IP and two-hybrid in human cells\",\n      \"pmids\": [\"10783164\", \"11095689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MCM10 acts before or after pre-RC formation unresolved at this stage\", \"Human interaction data from single lab\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Placed MCM10 epistatically between pre-RC formation and Cdc45 loading and revealed a role in DDK-dependent MCM phosphorylation, refining its temporal position in initiation.\",\n      \"evidence\": \"Degron inactivation and chromatin assays in S. pombe; in vitro kinase assay with Dfp1-Hsk1 and truncation mapping; two-hybrid/RNAi in Drosophila\",\n      \"pmids\": [\"12972571\", \"12604790\", \"12808023\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration that MCM10 stimulates unwinding not yet available\", \"Kinase-stimulation mechanism unresolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined two distinct activities: facilitating Cdc45 recruitment to origins and stabilizing the pol-alpha catalytic subunit against degradation.\",\n      \"evidence\": \"ChIP, co-IP, overexpression suppression and conditional degron in S. cerevisiae\",\n      \"pmids\": [\"15201046\", \"15494305\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of pol-alpha stabilization not yet defined\", \"Cdc45 interaction surface unmapped\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Assigned the pol-alpha-stabilizing activity to a conserved Hsp10-like chaperone domain and linked MCM10 to PCNA via a PIP box dependent on diubiquitination.\",\n      \"evidence\": \"Domain mutagenesis with rescue and PIP-box mutational analysis with genetic rescue in S. cerevisiae\",\n      \"pmids\": [\"16675460\", \"16782870\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the ubiquitin ligase for MCM10 diubiquitination unknown\", \"Functional consequence of PCNA binding for Okazaki processing not directly tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved MCM10 domain architecture and DNA-/pol-alpha-binding surfaces, and integrated MCM10 into the And-1/Ctf4 axis for pol-alpha chromatin loading.\",\n      \"evidence\": \"Crystallography, NMR and biochemistry of Xenopus Mcm10 domains; EM of human hexamer; co-IP and Xenopus extract depletion for And-1; siRNA in HeLa for human p180 stabilization\",\n      \"pmids\": [\"19081065\", \"18065420\", \"17823614\", \"17761813\", \"17699597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hexameric EM structure lacks mutagenesis validation\", \"Stoichiometry on DNA in vivo unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established the structural handoff logic (ssDNA and pol-alpha compete for the ID OB-fold) and embedded MCM10 in CMG/RECQ4 helicase complex assembly in human cells.\",\n      \"evidence\": \"Crystallography of ID-ssDNA complex with competition assays; purified complex co-IP and in vitro unwinding for RECQ4; BiFC and siRNA for CMG formation; EMSA/SPR for cooperative DNA binding\",\n      \"pmids\": [\"19608746\", \"19696745\", \"19805216\", \"19605346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Timing of pol-alpha handoff relative to unwinding unresolved\", \"RECQ4-MCM10 functional hierarchy not yet ordered\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Three independent acute-depletion studies converged on a discrete post-CMG-assembly function: MCM10 is required for origin DNA unwinding, CMG translocation, and RPA loading, not for CMG assembly itself.\",\n      \"evidence\": \"Auxin-inducible and off-AID degron systems with ChIP and 2D gels in S. cerevisiae and S. pombe\",\n      \"pmids\": [\"22433841\", \"22433840\", \"22285032\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism of the unwinding step not yet reconstituted\", \"Role of the zinc finger in RPA loading mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapped the MCM10-MCM2-7 interface to the C-terminus contacting Mcm2/Mcm6 (overlapping the Cdt1 site), distinguished low- versus high-affinity recruitment modes, and tied MCM10 to DDK stimulation and Cdc45/GINS stabilization.\",\n      \"evidence\": \"Pulldowns with purified MCM double/single hexamers, replication reconstitution, endogenous DH purification with fusion rescue, and in vitro DDK assays in S. cerevisiae; ChIP and siRNA for RecQL4-dependent recruitment in human cells\",\n      \"pmids\": [\"26719337\", \"26686640\", \"26582917\", \"25602958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How MCM10 promotes double-hexamer remodeling structurally undefined\", \"Order of RecQL4 and MCM10 origin loading still debated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Reconstitution demonstrated that MCM10 stimulates CMG helicase activity, supports elongation and isomerizes CMG to a strand-exclusion mode, with separation-of-function alleles uncoupling initial unwinding from elongation/CMG binding.\",\n      \"evidence\": \"In vitro replication and helicase/roadblock-bypass assays with purified CMG and Mcm10, mutagenesis and AID depletion in S. cerevisiae\",\n      \"pmids\": [\"28270517\", \"28869037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the duplex-to-strand-exclusion isomerization unresolved\", \"Contribution to lagging-strand engagement in vivo not fully defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined MCM10's strand-annealing activity and its physical footprint on six CMG subunits at the fork junction, linking it to suppression of SMARCAL1-driven fork regression.\",\n      \"evidence\": \"In vitro strand-annealing and fork-regression assays with purified SMARCAL1, plus cross-linking mass spectrometry of CMG-Mcm10\",\n      \"pmids\": [\"30598452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of CMG-Mcm10 lacking\", \"In vivo relevance of fork-regression inhibition not directly tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected MCM10 to genome- and telomere-protective functions, including BRCA2-dependent suppression of PRIMPOL repriming and prevention of telomere erosion under replication stress.\",\n      \"evidence\": \"Co-IP, siRNA and ssDNA-gap/fiber assays for BRCA2-PRIMPOL; patient fibroblasts, CRISPR/iPSC modeling, telomere FISH and MUS81 epistasis for telomere phenotypes\",\n      \"pmids\": [\"34645815\", \"33712616\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration that MCM10 disease mutations drive telomere defects in patients limited\", \"Mechanism linking strand-annealing to PRIMPOL suppression not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended MCM10 function to replisome processivity and repeat-tract stability, showing it stabilizes stalled replisomes and prevents fork stalling and instability at structured repeats.\",\n      \"evidence\": \"Single-molecule SV40 replisome reconstitution with human Mcm10; repeat-instability assays and live-cell fork-stalling imaging in S. cerevisiae\",\n      \"pmids\": [\"38967018\", \"39627228\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SV40 is a heterologous system\", \"Repeat-instability mechanism dissected in single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MCM10's multiple in vitro activities (origin melting, CMG isomerization, strand annealing, pol-alpha handoff, RECQ4 cooperation) are temporally coordinated and structurally integrated at the firing origin remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of the MCM10-CMG-origin intermediate\", \"Order of RECQ4 versus MCM10 loading not definitively settled\", \"Mechanism coupling unwinding to CMG translocation undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [14, 15, 18, 19, 40, 46]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [14, 18]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [10, 12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [11, 18, 32, 34]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [16, 34, 36, 37]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 4, 58]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [20, 21, 33, 55]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [3, 23, 32, 36, 37]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [20, 25, 34]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [35, 43, 44]}\n    ],\n    \"complexes\": [\"CMG helicase\", \"MCM2-7 double hexamer\"],\n    \"partners\": [\"MCM2\", \"MCM6\", \"CDC45\", \"RECQ4\", \"WDHD1\", \"POLA1\", \"PCNA\", \"BRCA2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":10,"faith_total":10,"faith_pct":100.0}}