{"gene":"ORC6","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1993,"finding":"ORC6 encodes the 50 kDa subunit of the yeast origin recognition complex (ORC) and interacts in vivo with yeast replication origins, establishing ORC as an in vivo origin-binding complex.","method":"One-hybrid screen; peptide sequencing of purified ORC subunit","journal":"Science","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — original identification via one-hybrid assay plus direct peptide sequencing, foundational replicated result","pmids":["8266075"],"is_preprint":false},{"year":2000,"finding":"Human ORC6 (hsORC6) does not co-immunoprecipitate stoichiometrically with ORC2-5 subunits, suggesting a more peripheral association with the human ORC holocomplex compared to yeast; it localizes to the nucleus along with other ORC subunits, and co-immunoprecipitates with a 65 kDa protein hyperphosphorylated in G1 and dephosphorylated in mitosis.","method":"Co-immunoprecipitation; cell fractionation; Western blot across cell cycle stages","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reciprocal co-IP and fractionation, single lab, two orthogonal methods","pmids":["10945994"],"is_preprint":false},{"year":2002,"finding":"Human Orc6 localizes to kinetochores and a reticular-like structure at the cell periphery during mitosis, and to the midbody before cytokinesis; siRNA-mediated depletion causes multipolar spindles, aberrant mitosis, multinucleated cells, and decreased DNA replication, demonstrating essential roles in chromosome segregation and cytokinesis.","method":"Immunofluorescence microscopy; siRNA knockdown; flow cytometry","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean siRNA KD with multiple defined cellular phenotypes plus live localization, replicated by independent later studies","pmids":["12169736"],"is_preprint":false},{"year":2003,"finding":"Drosophila Orc6 localizes to the cell membrane and cleavage furrow during cell division via its distinct C-terminal domain; this domain mediates interaction with the septin protein Pnut, as shown by two-hybrid and co-immunoprecipitation. Deletion of this C-terminal domain abolishes membrane/furrow localization and causes multinucleated cells without impairing DNA replication, demonstrating that cytokinesis and replication functions reside in separable domains.","method":"Two-hybrid screen; co-immunoprecipitation; immunofluorescence; dsRNA knockdown; deletion mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus mutagenesis plus localization with functional consequence, replicated in later studies","pmids":["12878722"],"is_preprint":false},{"year":2004,"finding":"The S-phase cyclin Clb5 binds directly and stably to yeast ORC via an RXL/Cy motif in the Orc6 subunit, recognized by the hydrophobic patch of Clb5; this interaction is maintained from S phase through M phase and functions to prevent reinitiation at replicated origins (replication control switch), not for replication initiation per se.","method":"In vitro binding assay; site-directed mutagenesis of Cy motif; genetic epistasis; overreplication assay","journal":"Genes & Development","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro binding plus mutagenesis of docking motif plus genetic epistasis, single lab but multiple orthogonal methods","pmids":["15105375"],"is_preprint":false},{"year":2006,"finding":"In budding yeast, Orc6 is required for DNA replication entry into S phase after pre-RC formation; depletion in late G1 displaces Mcm2 and Mcm10 from chromatin and severely reduces replication origin firing. Orc6-YFP shows a punctate nuclear pattern consistent with subnuclear replication foci; no mitotic or cytokinetic function was detected in yeast.","method":"Conditional depletion; chromatin fractionation; live-cell imaging (YFP); DNA combing/BrdU incorporation","journal":"The EMBO Journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KD at defined cell cycle stages with multiple readouts (chromatin fractionation, DNA combing, imaging), single lab","pmids":["17053779"],"is_preprint":false},{"year":2007,"finding":"Drosophila Orc6 directly binds DNA—preferring poly(dA) sequences including replication origin fragments—via its N-terminal core replication domain (excluding the C-terminal domain); mutations in this domain abolish ORC DNA binding and DNA replication in vitro, and prevent chromosome association and cause dominant-negative effects in vivo.","method":"In vitro DNA binding assay; reconstituted Drosophila ORC replication assay; site-directed mutagenesis; chromatin immunoprecipitation/chromosome spreading in vivo","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro replication assay plus mutagenesis plus in vivo chromatin association, single lab","pmids":["17283052"],"is_preprint":false},{"year":2007,"finding":"Yeast Orc6 recruits Cdt1 through two direct binding regions and its C-terminus (Orc6-CTD) anchors it to the Orc1-5 subcomplex; ORC lacking Orc6 fails to interact with Cdt1 or load Mcm2-7 onto origin DNA. A Cdt1–Orc6-CTD fusion rescues single-round but not multiple rounds of Mcm2-7 loading, demonstrating that dynamic Cdt1–Orc6 association is required for iterative helicase loading.","method":"In vitro Mcm2-7 loading assay; direct binding assay; reconstituted fusion protein complementation; yeast genetics","journal":"Genes & Development","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro loading assay plus direct binding plus genetic complementation, strong mechanistic resolution","pmids":["18006685"],"is_preprint":false},{"year":2008,"finding":"Drosophila Orc6 directly binds the septin complex (purified from embryos or reconstituted from recombinant proteins) via the coiled-coil domain of Pnut; Orc6 binding increases the intrinsic GTPase activity of the septin complex and, in the absence of GTP, enhances septin filament formation.","method":"Septin complex purification; recombinant reconstitution; GTPase activity assay; electron microscopy of filaments","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution with enzymatic assay and structural visualization, single lab but multiple orthogonal methods","pmids":["18987337"],"is_preprint":false},{"year":2009,"finding":"In Drosophila, the N-terminal domain of Orc6 mediates the DNA replication function while the C-terminal domain is required for passage through M phase; deletion of C-terminal domain releases G1 arrest and restores DNA replication but causes mitotic accumulation. Human Orc6 rescues DNA replication in Drosophila orc6 deletion cells, demonstrating cross-species conservation of the replication function.","method":"P-element excision (orc6 deletion); transgenic rescue with deletion/point mutants; cell cycle analysis; cross-species complementation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo genetic dissection with domain-specific mutants and cross-species rescue, single lab","pmids":["19541634"],"is_preprint":false},{"year":2011,"finding":"The middle domain of human Orc6 adopts a fold homologous to the helical domain of transcription factor TFIIB; mutagenesis of residues identified by this structure abolishes DNA binding by Orc6 and reduces DNA replication in vitro and in cultured cells, defining Orc6 as a DNA-binding subunit of metazoan ORC.","method":"X-ray crystallography (structure determination); site-directed mutagenesis; in vitro DNA binding assay; in vitro replication assay; cell-based replication assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vitro and cell-based functional validation, single lab but multiple orthogonal methods","pmids":["21502537"],"is_preprint":false},{"year":2011,"finding":"Human Orc6 interacts with Cdc6 (co-immunoprecipitation); this interaction is required for licensing DNA replication (pre-RC formation). Orc6 also interacts with the chromatin chaperone HMGA1a via its acidic C-terminus and AT-hooks, potentially directing ORC to AT-rich heterochromatic origins.","method":"Co-immunoprecipitation; imaging; chromatin recruitment assay; domain-deletion analysis","journal":"Cellular and Molecular Life Sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus imaging-based licensing assay plus domain mapping, single lab","pmids":["21461783"],"is_preprint":false},{"year":2011,"finding":"Using a temperature-sensitive N-end rule degron of avian Orc6, acute depletion specifically during mitosis (not S phase) causes asymmetric division and failure of cytokinesis with delayed daughter cell abscission; the C-terminal 25 residues of Orc6 are required for this function. S-phase depletion causes centrosome amplification that is suppressed by G2 checkpoint inhibition, indicating it is an indirect replication-stress consequence.","method":"N-end rule degron (temperature-sensitive degradation); fluorescence bleaching (FRAP-based abscission assay); C-terminal deletion mutant rescue","journal":"The Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — acute cell-cycle-stage-specific depletion with defined molecular mutant rescue and functional assay, single lab","pmids":["21422227"],"is_preprint":false},{"year":2013,"finding":"Cryo-EM analysis shows metazoan ORC adopts a global architecture similar to budding yeast ORC. A Meier-Gorlin syndrome mutation in the conserved C-terminal helix of Orc6 impedes recruitment of Orc6 into the ORC hexamer; biochemical studies show this C-terminal region of Orc6 binds a previously uncharacterized domain of Orc3, and this interaction is required for ORC function and MCM2-7 loading in vivo.","method":"3D electron microscopy; bioinformatic structural analysis; biochemical binding assay; in vivo MCM loading assay; site-directed mutagenesis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure plus direct biochemical binding plus in vivo functional assay, single study with multiple orthogonal methods","pmids":["24137536"],"is_preprint":false},{"year":2014,"finding":"Drosophila Orc6 forms dimers through interactions of its N-terminal TFIIB-like domains and directly binds the septin complex to facilitate septin filament formation; Orc6 acts as a molecular bridge stimulating septin polymerization. GTP-binding/hydrolysis by Pnut, Sep1, and Sep2, and intact C-terminal domains of septins, are required for complex integrity.","method":"Recombinant septin complex reconstitution; in vitro filament formation assay; mutagenesis of GTP-binding domains; biochemical binding assay","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro system with mutagenesis and functional filament assay, single lab","pmids":["25355953"],"is_preprint":false},{"year":2017,"finding":"Recruitment of ORC6L (Orc6) from a dormant maternal mRNA via a CPE element in its 3' UTR during mouse oocyte maturation is required for DNA replication in 1-cell embryos; RNAi ablation of the maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and blocks DNA replication after fertilization.","method":"RNAi-mediated maternal mRNA ablation; Western blot; DNA replication assay in embryos","journal":"Developmental Biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional KD with defined molecular and cellular readout, single lab","pmids":["20219456"],"is_preprint":false},{"year":2020,"finding":"Solution NMR structure of full-length human Orc6 reveals three independent domains (N, M, C); a DNA-binding domain (HsOrc6-DBD) within these domains is identified; mutagenesis of key residues abolishes DNA binding and reduces DNA replication, confirming Orc6 as a DNA-binding subunit of human ORC.","method":"Solution NMR; mutagenesis; in vitro DNA binding assay; cell-based DNA replication assay","journal":"Nucleic Acids Research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure plus mutagenesis plus functional validation in vitro and in cells, single lab","pmids":["32986843"],"is_preprint":false},{"year":2022,"finding":"Human Orc6 localizes to the replication fork during S phase and functions as an accessory factor for the mismatch repair (MMR) complex; Orc6 directly binds MutSα and enhances chromatin association of MutLα; without Orc6, MMR complex assembly and checkpoint signaling in response to oxidative DNA damage are abrogated.","method":"Co-immunoprecipitation (Orc6–MutSα); chromatin fractionation (MutLα association); replication fork localization (iPOND or equivalent); MMR activity assay; checkpoint signaling assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct binding assay plus chromatin fractionation plus functional MMR assay, single lab with multiple orthogonal methods","pmids":["35622890"],"is_preprint":false},{"year":2023,"finding":"Human Orc6 is phosphorylated at Thr229 predominantly during S phase in response to oxidative stress; this ATR-dependent phosphorylation is required for DNA damage checkpoint signaling (ATR signaling), fork progression halting, and efficient repair to prevent tumorigenesis. Phospho-dead Orc6 increases tumorigenicity.","method":"Phospho-specific antibody; site-directed mutagenesis (T229A phospho-dead); ATR signaling assay; cell proliferation/tumorigenicity assay","journal":"Molecular and Cellular Biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-specific mutagenesis plus checkpoint signaling assay plus tumorigenicity, single lab","pmids":["37096556"],"is_preprint":false},{"year":2024,"finding":"CDK-dependent phosphorylation of human Orc6 at Thr195 occurs during mitosis; the phosphomimetic T195E mutant impedes S-phase progression. Phosphorylated Orc6 associates more robustly with ORC outside G1, suggesting phospho-Orc6 prevents licensing activity of Orc1-5 outside G1. Orc6 and phospho-Orc6 localize to nucleolar organizing centers and regulate ribosome biogenesis.","method":"Site-directed mutagenesis (T195E phosphomimetic); co-immunoprecipitation; cell cycle analysis; nucleolar localization (immunofluorescence); ribosome biogenesis assay","journal":"Molecular and Cellular Biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — phosphomimetic mutagenesis with multiple functional readouts and co-IP, single lab","pmids":["38867464"],"is_preprint":false},{"year":2025,"finding":"During ORC binding-site switching in replication origin licensing, the N-terminal half of Orc6 (folded Orc6N domain plus adjacent unstructured linker) tethers ORC to the N-terminal region of Mcm2, preventing ORC release into solution; this tethering precedes ORC release from initial Mcm2-7 binding and is required for efficient double-hexamer formation. CDK phosphorylation of ORC inhibits this Orc6-Mcm2 tethering interaction, providing a mechanism for CDK inhibition of MCM loading.","method":"Single-molecule FRET assay; mutagenesis of Orc6 linker; in vitro MCM loading assay; CDK phosphorylation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — single-molecule FRET plus mutagenesis plus in vitro reconstituted MCM loading assay, single lab with multiple orthogonal methods","pmids":["41055997"],"is_preprint":false},{"year":2025,"finding":"Human Orc6 dissociates from chromatin upon S-phase entry in a proteasome-dependent manner; inhibition of the proteasome causes accumulation of chromatin-bound Orc6, which promotes aberrant MCM loading after S-phase entry, ultimately leading to tetraploid cell formation.","method":"Chromatin fractionation; proteasome inhibitor treatment; MCM loading assay; cell cycle/ploidy analysis (flow cytometry)","journal":"Journal of Cell Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chromatin fractionation plus functional MCM loading plus ploidy readout with pharmacological intervention, single lab","pmids":["40554748"],"is_preprint":false},{"year":2024,"finding":"ORC6 associates with nuclear p65 after LPS stimulation; this interaction is necessary for NFκB activation in macrophages. ORC6 silencing or knockout inhibits LPS-induced NFκB activation and pro-inflammatory cytokine production, while ORC6 overexpression enhances these responses and cannot rescue the response when p65 is silenced.","method":"Co-immunoprecipitation (ORC6–p65); CRISPR/Cas9 knockout; shRNA silencing; cytokine ELISA; NFκB reporter assay; in vivo macrophage-specific knockdown","journal":"Cell Communication and Signaling","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus KO plus OE with epistasis experiment (p65 silencing), single lab","pmids":["39143485"],"is_preprint":false}],"current_model":"ORC6 encodes the smallest and least conserved subunit of the origin recognition complex (ORC), which binds replication origins in vivo; Orc6 directly contacts Cdt1 to enable iterative Mcm2-7 helicase loading during pre-RC formation, tethers ORC to Mcm2 via its N-terminal domain during the binding-site switch required for double-hexamer assembly, and its proteasome-mediated removal from chromatin at S-phase entry prevents inappropriate MCM reloading. In metazoans, Orc6 has an additional, separable C-terminal domain that interacts with septins (stimulating GTPase activity and filament formation) and is required for cytokinesis/abscission, while its TFIIB-like middle/N-terminal domains mediate DNA binding at origins. Human Orc6 is phosphorylated by CDK at T195 (mitosis, inhibiting re-licensing) and by ATR at T229 (S-phase, enabling mismatch repair complex assembly and DNA damage checkpoint signaling), and associates with nuclear p65 to facilitate NFκB activation."},"narrative":{"mechanistic_narrative":"ORC6 encodes the smallest subunit of the origin recognition complex (ORC), an in vivo origin-binding complex first defined in budding yeast [PMID:8266075], and functions as a DNA-binding, helicase-loading subunit essential for the licensing of replication origins [PMID:18006685, PMID:21502537]. Its middle domain adopts a TFIIB-like fold, and structural and mutagenesis studies of both the human protein and the Drosophila ortholog define Orc6 as a sequence-selective DNA-binding subunit of metazoan ORC that prefers poly(dA)/origin DNA and is required for replication in vitro and in cells [PMID:17283052, PMID:21502537, PMID:32986843]. Orc6 anchors itself into the holocomplex through C-terminal contacts—to the Orc1-5 subcomplex in yeast and to a domain of Orc3 in metazoans, an interaction disrupted by a Meier-Gorlin syndrome mutation that impairs Orc6 incorporation and MCM2-7 loading [PMID:18006685, PMID:24137536]. Mechanistically, Orc6 recruits Cdt1 through direct binding to enable iterative Mcm2-7 loading, and its N-terminal domain plus adjacent linker tethers ORC to Mcm2 during the binding-site switch that builds the MCM double hexamer; CDK phosphorylation of ORC inhibits this tethering, linking helicase loading to cell-cycle control [PMID:18006685, PMID:41055997]. Re-licensing is further blocked by CDK phosphorylation of human Orc6 at Thr195 and by proteasome-dependent removal of Orc6 from chromatin at S-phase entry, which prevents aberrant MCM reloading and tetraploidy [PMID:38867464, PMID:40554748]. In metazoans Orc6 carries a separable C-terminal cytokinesis function: it binds the septin Pnut/septin complex, stimulating septin GTPase activity and filament formation, and is required for furrow/midbody localization and abscission, such that domain deletion uncouples cytokinesis defects from intact DNA replication [PMID:12169736, PMID:12878722, PMID:18987337, PMID:25355953]. Beyond core replication, human Orc6 localizes to the replication fork and acts as an accessory factor for mismatch repair by binding MutSα and promoting MutLα chromatin association, with ATR-dependent phosphorylation at Thr229 driving oxidative-damage checkpoint signaling [PMID:35622890, PMID:37096556]. ORC6 also associates with nuclear p65 to support LPS-induced NFκB activation in macrophages [PMID:39143485].","teleology":[{"year":1993,"claim":"Established that ORC6 is a bona fide subunit of an origin-binding complex, defining ORC as the in vivo recognition machinery for replication origins.","evidence":"One-hybrid screen and peptide sequencing of the purified 50 kDa yeast ORC subunit","pmids":["8266075"],"confidence":"High","gaps":["Did not resolve Orc6's specific molecular contribution within ORC","No structural or DNA-contact information"]},{"year":2002,"claim":"Revealed that metazoan Orc6 has roles beyond replication, localizing to mitotic structures and being required for chromosome segregation and cytokinesis.","evidence":"Immunofluorescence, siRNA depletion and flow cytometry in human cells","pmids":["12169736"],"confidence":"High","gaps":["Did not identify the molecular partners mediating the cytokinesis function","Did not separate replication from division defects"]},{"year":2003,"claim":"Mapped the cytokinesis function to a separable C-terminal domain that interacts with the septin Pnut, distinguishing it from the replication function.","evidence":"Two-hybrid, co-IP, deletion mutagenesis and dsRNA knockdown in Drosophila","pmids":["12878722"],"confidence":"High","gaps":["Did not establish the biochemical effect of Orc6 on septins","Mammalian conservation of the septin interaction untested here"]},{"year":2004,"claim":"Showed Orc6 carries an RXL/Cy motif that docks S-phase cyclin Clb5, defining a mechanism by which Orc6 enforces a re-replication control switch rather than initiation.","evidence":"In vitro binding, Cy-motif mutagenesis, genetic epistasis and overreplication assays in yeast","pmids":["15105375"],"confidence":"High","gaps":["Did not address whether metazoan Orc6 uses an analogous cyclin docking","Mechanism of overreplication suppression at origins not fully resolved"]},{"year":2007,"claim":"Identified Orc6 as a direct DNA-binding subunit, localizing origin recognition activity to its N-terminal core domain.","evidence":"In vitro DNA binding, reconstituted Drosophila ORC replication assay, mutagenesis and in vivo chromatin association","pmids":["17283052"],"confidence":"High","gaps":["Structural basis of DNA recognition not defined here","Sequence specificity determinants only partially mapped"]},{"year":2007,"claim":"Defined Orc6 as the Cdt1-recruiting subunit required for iterative Mcm2-7 loading, mechanistically linking it to helicase loading.","evidence":"Reconstituted in vitro Mcm2-7 loading, direct binding, fusion complementation and yeast genetics","pmids":["18006685"],"confidence":"High","gaps":["Did not visualize the structural transitions during loading","Did not establish how Orc6-Cdt1 cycling is regulated"]},{"year":2009,"claim":"Genetically separated the N-terminal replication function from the C-terminal mitotic function and demonstrated cross-species conservation of the replication role.","evidence":"P-element deletion, domain-mutant transgenic rescue and human-to-fly complementation in Drosophila","pmids":["19541634"],"confidence":"High","gaps":["Did not define how the two domains are coordinated in vivo","Mitotic mechanism still unresolved at this stage"]},{"year":2011,"claim":"Provided a structural explanation for Orc6 DNA binding, showing its middle domain folds like the TFIIB helical domain.","evidence":"X-ray crystallography, mutagenesis and in vitro/cell-based replication assays for human Orc6","pmids":["21502537"],"confidence":"High","gaps":["Structure of full-length Orc6 not solved here","DNA-bound complex structure not determined"]},{"year":2011,"claim":"Extended the metazoan pre-RC interaction map, implicating Orc6 contacts with Cdc6 and the chromatin chaperone HMGA1a in origin licensing and targeting.","evidence":"Co-IP, imaging, chromatin recruitment and domain-deletion analysis in human cells","pmids":["21461783"],"confidence":"Medium","gaps":["Interactions shown by co-IP without reciprocal in vitro reconstitution","Functional contribution of HMGA1a-directed targeting not quantified"]},{"year":2011,"claim":"Used acute cell-cycle-stage-specific depletion to prove the cytokinesis/abscission role is direct and resides in the C-terminal 25 residues, separating it from replication-stress phenotypes.","evidence":"N-end rule degron, FRAP-based abscission assay and C-terminal deletion rescue in avian cells","pmids":["21422227"],"confidence":"High","gaps":["Molecular effectors at the midbody not fully defined","Did not connect to septin biochemistry directly in this system"]},{"year":2008,"claim":"Demonstrated the biochemical consequence of the Orc6-septin interaction—stimulating septin GTPase activity and filament assembly.","evidence":"Septin purification, recombinant reconstitution, GTPase assay and electron microscopy of filaments","pmids":["18987337"],"confidence":"High","gaps":["In vivo relevance of GTPase stimulation during cytokinesis not directly tested","Did not resolve binding stoichiometry"]},{"year":2013,"claim":"Placed Orc6 in the metazoan ORC architecture and tied a Meier-Gorlin syndrome mutation to defective Orc6-Orc3 assembly and MCM loading.","evidence":"3D electron microscopy, biochemical binding, in vivo MCM loading and mutagenesis","pmids":["24137536"],"confidence":"High","gaps":["High-resolution structure of the Orc6-Orc3 interface not obtained","Did not survey other disease alleles"]},{"year":2014,"claim":"Refined the cytokinesis mechanism by showing Orc6 dimerizes via TFIIB-like domains and bridges septins to promote filament formation.","evidence":"Recombinant septin reconstitution, in vitro filament assay and GTP-binding-domain mutagenesis in Drosophila","pmids":["25355953"],"confidence":"High","gaps":["Did not determine the bridging structure at atomic resolution","Connection between dimerization and replication function unclear"]},{"year":2020,"claim":"Determined the full-length human Orc6 three-domain solution structure and localized a discrete DNA-binding domain confirming its origin-binding role.","evidence":"Solution NMR, mutagenesis and in vitro/cell-based replication assays","pmids":["32986843"],"confidence":"High","gaps":["DNA-bound conformation not captured","Domain interplay within intact ORC not resolved"]},{"year":2017,"claim":"Established a developmental requirement for translational activation of maternal Orc6 mRNA for the first embryonic replication.","evidence":"CPE-targeted RNAi maternal mRNA ablation, Western blot and embryo replication assay in mouse","pmids":["20219456"],"confidence":"Medium","gaps":["Translational control machinery only inferred from the CPE element","Single-system functional readout"]},{"year":2022,"claim":"Uncovered a non-replication chromatin role: Orc6 at the replication fork acts as an accessory factor for mismatch repair by binding MutSα and promoting MutLα loading.","evidence":"Co-IP, chromatin fractionation, fork localization and MMR/checkpoint assays in human cells","pmids":["35622890"],"confidence":"High","gaps":["Structural basis of Orc6-MutSα binding undefined","Whether this is separable from origin licensing not fully resolved"]},{"year":2023,"claim":"Identified ATR-dependent Thr229 phosphorylation of Orc6 as the trigger for oxidative-damage checkpoint signaling and tumor suppression.","evidence":"Phospho-specific antibody, T229A mutagenesis, ATR signaling and tumorigenicity assays","pmids":["37096556"],"confidence":"Medium","gaps":["Direct ATR-Orc6 kinase relationship inferred from dependency","Downstream effectors of phospho-Orc6 not fully mapped"]},{"year":2024,"claim":"Defined a CDK-dependent Thr195 phosphorylation that restrains re-licensing outside G1 and linked Orc6 to nucleolar function and ribosome biogenesis.","evidence":"T195E phosphomimetic mutagenesis, co-IP, cell cycle analysis and ribosome biogenesis assays","pmids":["38867464"],"confidence":"Medium","gaps":["Phosphomimetic does not fully recapitulate endogenous phosphorylation","Mechanism connecting Orc6 to ribosome biogenesis unresolved"]},{"year":2024,"claim":"Implicated Orc6 in innate immune signaling through a p65 interaction required for LPS-induced NFκB activation.","evidence":"Co-IP, CRISPR knockout, shRNA silencing, cytokine and NFκB reporter assays with p65-epistasis in macrophages","pmids":["39143485"],"confidence":"Medium","gaps":["Direct versus indirect Orc6-p65 binding not structurally defined","Relationship to Orc6 replication function unknown"]},{"year":2025,"claim":"Resolved how Orc6 enforces double-hexamer assembly by tethering ORC to Mcm2 during the binding-site switch, and how CDK phosphorylation disrupts this to inhibit loading.","evidence":"Single-molecule FRET, Orc6 linker mutagenesis, in vitro MCM loading and CDK phosphorylation assays","pmids":["41055997"],"confidence":"High","gaps":["Precise CDK target residues on ORC controlling tethering not all defined","In vivo confirmation of the tethering intermediate pending"]},{"year":2025,"claim":"Showed proteasome-dependent removal of Orc6 from chromatin at S-phase entry is a safeguard against MCM reloading and tetraploidy.","evidence":"Chromatin fractionation, proteasome inhibition, MCM loading and ploidy analysis in human cells","pmids":["40554748"],"confidence":"Medium","gaps":["Ubiquitin ligase mediating Orc6 turnover not identified","Direct evidence Orc6 is the proteasome substrate versus indirect effect not fully separated"]},{"year":null,"claim":"How Orc6's distinct activities—origin DNA binding, helicase loading, septin/cytokinesis, mismatch repair, checkpoint signaling, nucleolar/ribosome biogenesis and NFκB signaling—are spatially and temporally coordinated within a single small protein remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating replication and non-replication functions","Regulatory hierarchy among the multiple phosphorylation events undefined","Structure of Orc6 bound simultaneously to DNA and partner proteins lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6,10,16]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[8,14]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[6,21]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[19]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[7,10,20]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,12,19]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[17,18]}],"complexes":["origin recognition complex (ORC)","septin complex"],"partners":["CDT1","MCM2","ORC3","CDC6","PNUT","MUTSΑ","RELA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y5N6","full_name":"Origin recognition complex subunit 6","aliases":[],"length_aa":252,"mass_kda":28.1,"function":"Component of the origin recognition complex (ORC) that binds origins of replication. DNA-binding is ATP-dependent. The specific DNA sequences that define origins of replication have not been identified yet. ORC is required to assemble the pre-replication complex necessary to initiate DNA replication. Does not bind histone H3 and H4 trimethylation marks H3K9me3, H3K27me3 and H4K20me3","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9Y5N6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ORC6","classification":"Common Essential","n_dependent_lines":1202,"n_total_lines":1208,"dependency_fraction":0.9950331125827815},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ORC6","total_profiled":1310},"omim":[{"mim_id":"613803","title":"MEIER-GORLIN SYNDROME 3; MGORS3","url":"https://www.omim.org/entry/613803"},{"mim_id":"607213","title":"ORIGIN RECOGNITION COMPLEX, SUBUNIT 6; ORC6","url":"https://www.omim.org/entry/607213"},{"mim_id":"601902","title":"ORIGIN RECOGNITION COMPLEX, SUBUNIT 1; ORC1","url":"https://www.omim.org/entry/601902"},{"mim_id":"224690","title":"MEIER-GORLIN SYNDROME 1; MGORS1","url":"https://www.omim.org/entry/224690"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli fibrillar center","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":9.7},{"tissue":"lymphoid 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\"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — original identification via one-hybrid assay plus direct peptide sequencing, foundational replicated result\",\n      \"pmids\": [\"8266075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Human ORC6 (hsORC6) does not co-immunoprecipitate stoichiometrically with ORC2-5 subunits, suggesting a more peripheral association with the human ORC holocomplex compared to yeast; it localizes to the nucleus along with other ORC subunits, and co-immunoprecipitates with a 65 kDa protein hyperphosphorylated in G1 and dephosphorylated in mitosis.\",\n      \"method\": \"Co-immunoprecipitation; cell fractionation; Western blot across cell cycle stages\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reciprocal co-IP and fractionation, single lab, two orthogonal methods\",\n      \"pmids\": [\"10945994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human Orc6 localizes to kinetochores and a reticular-like structure at the cell periphery during mitosis, and to the midbody before cytokinesis; siRNA-mediated depletion causes multipolar spindles, aberrant mitosis, multinucleated cells, and decreased DNA replication, demonstrating essential roles in chromosome segregation and cytokinesis.\",\n      \"method\": \"Immunofluorescence microscopy; siRNA knockdown; flow cytometry\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean siRNA KD with multiple defined cellular phenotypes plus live localization, replicated by independent later studies\",\n      \"pmids\": [\"12169736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Drosophila Orc6 localizes to the cell membrane and cleavage furrow during cell division via its distinct C-terminal domain; this domain mediates interaction with the septin protein Pnut, as shown by two-hybrid and co-immunoprecipitation. Deletion of this C-terminal domain abolishes membrane/furrow localization and causes multinucleated cells without impairing DNA replication, demonstrating that cytokinesis and replication functions reside in separable domains.\",\n      \"method\": \"Two-hybrid screen; co-immunoprecipitation; immunofluorescence; dsRNA knockdown; deletion mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus mutagenesis plus localization with functional consequence, replicated in later studies\",\n      \"pmids\": [\"12878722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The S-phase cyclin Clb5 binds directly and stably to yeast ORC via an RXL/Cy motif in the Orc6 subunit, recognized by the hydrophobic patch of Clb5; this interaction is maintained from S phase through M phase and functions to prevent reinitiation at replicated origins (replication control switch), not for replication initiation per se.\",\n      \"method\": \"In vitro binding assay; site-directed mutagenesis of Cy motif; genetic epistasis; overreplication assay\",\n      \"journal\": \"Genes & Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro binding plus mutagenesis of docking motif plus genetic epistasis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15105375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In budding yeast, Orc6 is required for DNA replication entry into S phase after pre-RC formation; depletion in late G1 displaces Mcm2 and Mcm10 from chromatin and severely reduces replication origin firing. Orc6-YFP shows a punctate nuclear pattern consistent with subnuclear replication foci; no mitotic or cytokinetic function was detected in yeast.\",\n      \"method\": \"Conditional depletion; chromatin fractionation; live-cell imaging (YFP); DNA combing/BrdU incorporation\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KD at defined cell cycle stages with multiple readouts (chromatin fractionation, DNA combing, imaging), single lab\",\n      \"pmids\": [\"17053779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Drosophila Orc6 directly binds DNA—preferring poly(dA) sequences including replication origin fragments—via its N-terminal core replication domain (excluding the C-terminal domain); mutations in this domain abolish ORC DNA binding and DNA replication in vitro, and prevent chromosome association and cause dominant-negative effects in vivo.\",\n      \"method\": \"In vitro DNA binding assay; reconstituted Drosophila ORC replication assay; site-directed mutagenesis; chromatin immunoprecipitation/chromosome spreading in vivo\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro replication assay plus mutagenesis plus in vivo chromatin association, single lab\",\n      \"pmids\": [\"17283052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Yeast Orc6 recruits Cdt1 through two direct binding regions and its C-terminus (Orc6-CTD) anchors it to the Orc1-5 subcomplex; ORC lacking Orc6 fails to interact with Cdt1 or load Mcm2-7 onto origin DNA. A Cdt1–Orc6-CTD fusion rescues single-round but not multiple rounds of Mcm2-7 loading, demonstrating that dynamic Cdt1–Orc6 association is required for iterative helicase loading.\",\n      \"method\": \"In vitro Mcm2-7 loading assay; direct binding assay; reconstituted fusion protein complementation; yeast genetics\",\n      \"journal\": \"Genes & Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro loading assay plus direct binding plus genetic complementation, strong mechanistic resolution\",\n      \"pmids\": [\"18006685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Drosophila Orc6 directly binds the septin complex (purified from embryos or reconstituted from recombinant proteins) via the coiled-coil domain of Pnut; Orc6 binding increases the intrinsic GTPase activity of the septin complex and, in the absence of GTP, enhances septin filament formation.\",\n      \"method\": \"Septin complex purification; recombinant reconstitution; GTPase activity assay; electron microscopy of filaments\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution with enzymatic assay and structural visualization, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18987337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In Drosophila, the N-terminal domain of Orc6 mediates the DNA replication function while the C-terminal domain is required for passage through M phase; deletion of C-terminal domain releases G1 arrest and restores DNA replication but causes mitotic accumulation. Human Orc6 rescues DNA replication in Drosophila orc6 deletion cells, demonstrating cross-species conservation of the replication function.\",\n      \"method\": \"P-element excision (orc6 deletion); transgenic rescue with deletion/point mutants; cell cycle analysis; cross-species complementation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic dissection with domain-specific mutants and cross-species rescue, single lab\",\n      \"pmids\": [\"19541634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The middle domain of human Orc6 adopts a fold homologous to the helical domain of transcription factor TFIIB; mutagenesis of residues identified by this structure abolishes DNA binding by Orc6 and reduces DNA replication in vitro and in cultured cells, defining Orc6 as a DNA-binding subunit of metazoan ORC.\",\n      \"method\": \"X-ray crystallography (structure determination); site-directed mutagenesis; in vitro DNA binding assay; in vitro replication assay; cell-based replication assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis plus in vitro and cell-based functional validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21502537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human Orc6 interacts with Cdc6 (co-immunoprecipitation); this interaction is required for licensing DNA replication (pre-RC formation). Orc6 also interacts with the chromatin chaperone HMGA1a via its acidic C-terminus and AT-hooks, potentially directing ORC to AT-rich heterochromatic origins.\",\n      \"method\": \"Co-immunoprecipitation; imaging; chromatin recruitment assay; domain-deletion analysis\",\n      \"journal\": \"Cellular and Molecular Life Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus imaging-based licensing assay plus domain mapping, single lab\",\n      \"pmids\": [\"21461783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Using a temperature-sensitive N-end rule degron of avian Orc6, acute depletion specifically during mitosis (not S phase) causes asymmetric division and failure of cytokinesis with delayed daughter cell abscission; the C-terminal 25 residues of Orc6 are required for this function. S-phase depletion causes centrosome amplification that is suppressed by G2 checkpoint inhibition, indicating it is an indirect replication-stress consequence.\",\n      \"method\": \"N-end rule degron (temperature-sensitive degradation); fluorescence bleaching (FRAP-based abscission assay); C-terminal deletion mutant rescue\",\n      \"journal\": \"The Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — acute cell-cycle-stage-specific depletion with defined molecular mutant rescue and functional assay, single lab\",\n      \"pmids\": [\"21422227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cryo-EM analysis shows metazoan ORC adopts a global architecture similar to budding yeast ORC. A Meier-Gorlin syndrome mutation in the conserved C-terminal helix of Orc6 impedes recruitment of Orc6 into the ORC hexamer; biochemical studies show this C-terminal region of Orc6 binds a previously uncharacterized domain of Orc3, and this interaction is required for ORC function and MCM2-7 loading in vivo.\",\n      \"method\": \"3D electron microscopy; bioinformatic structural analysis; biochemical binding assay; in vivo MCM loading assay; site-directed mutagenesis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure plus direct biochemical binding plus in vivo functional assay, single study with multiple orthogonal methods\",\n      \"pmids\": [\"24137536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila Orc6 forms dimers through interactions of its N-terminal TFIIB-like domains and directly binds the septin complex to facilitate septin filament formation; Orc6 acts as a molecular bridge stimulating septin polymerization. GTP-binding/hydrolysis by Pnut, Sep1, and Sep2, and intact C-terminal domains of septins, are required for complex integrity.\",\n      \"method\": \"Recombinant septin complex reconstitution; in vitro filament formation assay; mutagenesis of GTP-binding domains; biochemical binding assay\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro system with mutagenesis and functional filament assay, single lab\",\n      \"pmids\": [\"25355953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Recruitment of ORC6L (Orc6) from a dormant maternal mRNA via a CPE element in its 3' UTR during mouse oocyte maturation is required for DNA replication in 1-cell embryos; RNAi ablation of the maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and blocks DNA replication after fertilization.\",\n      \"method\": \"RNAi-mediated maternal mRNA ablation; Western blot; DNA replication assay in embryos\",\n      \"journal\": \"Developmental Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional KD with defined molecular and cellular readout, single lab\",\n      \"pmids\": [\"20219456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Solution NMR structure of full-length human Orc6 reveals three independent domains (N, M, C); a DNA-binding domain (HsOrc6-DBD) within these domains is identified; mutagenesis of key residues abolishes DNA binding and reduces DNA replication, confirming Orc6 as a DNA-binding subunit of human ORC.\",\n      \"method\": \"Solution NMR; mutagenesis; in vitro DNA binding assay; cell-based DNA replication assay\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus mutagenesis plus functional validation in vitro and in cells, single lab\",\n      \"pmids\": [\"32986843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human Orc6 localizes to the replication fork during S phase and functions as an accessory factor for the mismatch repair (MMR) complex; Orc6 directly binds MutSα and enhances chromatin association of MutLα; without Orc6, MMR complex assembly and checkpoint signaling in response to oxidative DNA damage are abrogated.\",\n      \"method\": \"Co-immunoprecipitation (Orc6–MutSα); chromatin fractionation (MutLα association); replication fork localization (iPOND or equivalent); MMR activity assay; checkpoint signaling assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay plus chromatin fractionation plus functional MMR assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35622890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Human Orc6 is phosphorylated at Thr229 predominantly during S phase in response to oxidative stress; this ATR-dependent phosphorylation is required for DNA damage checkpoint signaling (ATR signaling), fork progression halting, and efficient repair to prevent tumorigenesis. Phospho-dead Orc6 increases tumorigenicity.\",\n      \"method\": \"Phospho-specific antibody; site-directed mutagenesis (T229A phospho-dead); ATR signaling assay; cell proliferation/tumorigenicity assay\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-specific mutagenesis plus checkpoint signaling assay plus tumorigenicity, single lab\",\n      \"pmids\": [\"37096556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CDK-dependent phosphorylation of human Orc6 at Thr195 occurs during mitosis; the phosphomimetic T195E mutant impedes S-phase progression. Phosphorylated Orc6 associates more robustly with ORC outside G1, suggesting phospho-Orc6 prevents licensing activity of Orc1-5 outside G1. Orc6 and phospho-Orc6 localize to nucleolar organizing centers and regulate ribosome biogenesis.\",\n      \"method\": \"Site-directed mutagenesis (T195E phosphomimetic); co-immunoprecipitation; cell cycle analysis; nucleolar localization (immunofluorescence); ribosome biogenesis assay\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — phosphomimetic mutagenesis with multiple functional readouts and co-IP, single lab\",\n      \"pmids\": [\"38867464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"During ORC binding-site switching in replication origin licensing, the N-terminal half of Orc6 (folded Orc6N domain plus adjacent unstructured linker) tethers ORC to the N-terminal region of Mcm2, preventing ORC release into solution; this tethering precedes ORC release from initial Mcm2-7 binding and is required for efficient double-hexamer formation. CDK phosphorylation of ORC inhibits this Orc6-Mcm2 tethering interaction, providing a mechanism for CDK inhibition of MCM loading.\",\n      \"method\": \"Single-molecule FRET assay; mutagenesis of Orc6 linker; in vitro MCM loading assay; CDK phosphorylation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule FRET plus mutagenesis plus in vitro reconstituted MCM loading assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41055997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Human Orc6 dissociates from chromatin upon S-phase entry in a proteasome-dependent manner; inhibition of the proteasome causes accumulation of chromatin-bound Orc6, which promotes aberrant MCM loading after S-phase entry, ultimately leading to tetraploid cell formation.\",\n      \"method\": \"Chromatin fractionation; proteasome inhibitor treatment; MCM loading assay; cell cycle/ploidy analysis (flow cytometry)\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chromatin fractionation plus functional MCM loading plus ploidy readout with pharmacological intervention, single lab\",\n      \"pmids\": [\"40554748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ORC6 associates with nuclear p65 after LPS stimulation; this interaction is necessary for NFκB activation in macrophages. ORC6 silencing or knockout inhibits LPS-induced NFκB activation and pro-inflammatory cytokine production, while ORC6 overexpression enhances these responses and cannot rescue the response when p65 is silenced.\",\n      \"method\": \"Co-immunoprecipitation (ORC6–p65); CRISPR/Cas9 knockout; shRNA silencing; cytokine ELISA; NFκB reporter assay; in vivo macrophage-specific knockdown\",\n      \"journal\": \"Cell Communication and Signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus KO plus OE with epistasis experiment (p65 silencing), single lab\",\n      \"pmids\": [\"39143485\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ORC6 encodes the smallest and least conserved subunit of the origin recognition complex (ORC), which binds replication origins in vivo; Orc6 directly contacts Cdt1 to enable iterative Mcm2-7 helicase loading during pre-RC formation, tethers ORC to Mcm2 via its N-terminal domain during the binding-site switch required for double-hexamer assembly, and its proteasome-mediated removal from chromatin at S-phase entry prevents inappropriate MCM reloading. In metazoans, Orc6 has an additional, separable C-terminal domain that interacts with septins (stimulating GTPase activity and filament formation) and is required for cytokinesis/abscission, while its TFIIB-like middle/N-terminal domains mediate DNA binding at origins. Human Orc6 is phosphorylated by CDK at T195 (mitosis, inhibiting re-licensing) and by ATR at T229 (S-phase, enabling mismatch repair complex assembly and DNA damage checkpoint signaling), and associates with nuclear p65 to facilitate NFκB activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ORC6 encodes the smallest subunit of the origin recognition complex (ORC), an in vivo origin-binding complex first defined in budding yeast [#0], and functions as a DNA-binding, helicase-loading subunit essential for the licensing of replication origins [#7, #10]. Its middle domain adopts a TFIIB-like fold, and structural and mutagenesis studies of both the human protein and the Drosophila ortholog define Orc6 as a sequence-selective DNA-binding subunit of metazoan ORC that prefers poly(dA)/origin DNA and is required for replication in vitro and in cells [#6, #10, #16]. Orc6 anchors itself into the holocomplex through C-terminal contacts—to the Orc1-5 subcomplex in yeast and to a domain of Orc3 in metazoans, an interaction disrupted by a Meier-Gorlin syndrome mutation that impairs Orc6 incorporation and MCM2-7 loading [#7, #13]. Mechanistically, Orc6 recruits Cdt1 through direct binding to enable iterative Mcm2-7 loading, and its N-terminal domain plus adjacent linker tethers ORC to Mcm2 during the binding-site switch that builds the MCM double hexamer; CDK phosphorylation of ORC inhibits this tethering, linking helicase loading to cell-cycle control [#7, #20]. Re-licensing is further blocked by CDK phosphorylation of human Orc6 at Thr195 and by proteasome-dependent removal of Orc6 from chromatin at S-phase entry, which prevents aberrant MCM reloading and tetraploidy [#19, #21]. In metazoans Orc6 carries a separable C-terminal cytokinesis function: it binds the septin Pnut/septin complex, stimulating septin GTPase activity and filament formation, and is required for furrow/midbody localization and abscission, such that domain deletion uncouples cytokinesis defects from intact DNA replication [#2, #3, #8, #14]. Beyond core replication, human Orc6 localizes to the replication fork and acts as an accessory factor for mismatch repair by binding MutSα and promoting MutLα chromatin association, with ATR-dependent phosphorylation at Thr229 driving oxidative-damage checkpoint signaling [#17, #18]. ORC6 also associates with nuclear p65 to support LPS-induced NF\\u03baB activation in macrophages [#22].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established that ORC6 is a bona fide subunit of an origin-binding complex, defining ORC as the in vivo recognition machinery for replication origins.\",\n      \"evidence\": \"One-hybrid screen and peptide sequencing of the purified 50 kDa yeast ORC subunit\",\n      \"pmids\": [\"8266075\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve Orc6's specific molecular contribution within ORC\", \"No structural or DNA-contact information\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Revealed that metazoan Orc6 has roles beyond replication, localizing to mitotic structures and being required for chromosome segregation and cytokinesis.\",\n      \"evidence\": \"Immunofluorescence, siRNA depletion and flow cytometry in human cells\",\n      \"pmids\": [\"12169736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the molecular partners mediating the cytokinesis function\", \"Did not separate replication from division defects\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapped the cytokinesis function to a separable C-terminal domain that interacts with the septin Pnut, distinguishing it from the replication function.\",\n      \"evidence\": \"Two-hybrid, co-IP, deletion mutagenesis and dsRNA knockdown in Drosophila\",\n      \"pmids\": [\"12878722\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the biochemical effect of Orc6 on septins\", \"Mammalian conservation of the septin interaction untested here\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed Orc6 carries an RXL/Cy motif that docks S-phase cyclin Clb5, defining a mechanism by which Orc6 enforces a re-replication control switch rather than initiation.\",\n      \"evidence\": \"In vitro binding, Cy-motif mutagenesis, genetic epistasis and overreplication assays in yeast\",\n      \"pmids\": [\"15105375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address whether metazoan Orc6 uses an analogous cyclin docking\", \"Mechanism of overreplication suppression at origins not fully resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified Orc6 as a direct DNA-binding subunit, localizing origin recognition activity to its N-terminal core domain.\",\n      \"evidence\": \"In vitro DNA binding, reconstituted Drosophila ORC replication assay, mutagenesis and in vivo chromatin association\",\n      \"pmids\": [\"17283052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of DNA recognition not defined here\", \"Sequence specificity determinants only partially mapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined Orc6 as the Cdt1-recruiting subunit required for iterative Mcm2-7 loading, mechanistically linking it to helicase loading.\",\n      \"evidence\": \"Reconstituted in vitro Mcm2-7 loading, direct binding, fusion complementation and yeast genetics\",\n      \"pmids\": [\"18006685\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not visualize the structural transitions during loading\", \"Did not establish how Orc6-Cdt1 cycling is regulated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Genetically separated the N-terminal replication function from the C-terminal mitotic function and demonstrated cross-species conservation of the replication role.\",\n      \"evidence\": \"P-element deletion, domain-mutant transgenic rescue and human-to-fly complementation in Drosophila\",\n      \"pmids\": [\"19541634\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how the two domains are coordinated in vivo\", \"Mitotic mechanism still unresolved at this stage\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided a structural explanation for Orc6 DNA binding, showing its middle domain folds like the TFIIB helical domain.\",\n      \"evidence\": \"X-ray crystallography, mutagenesis and in vitro/cell-based replication assays for human Orc6\",\n      \"pmids\": [\"21502537\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length Orc6 not solved here\", \"DNA-bound complex structure not determined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended the metazoan pre-RC interaction map, implicating Orc6 contacts with Cdc6 and the chromatin chaperone HMGA1a in origin licensing and targeting.\",\n      \"evidence\": \"Co-IP, imaging, chromatin recruitment and domain-deletion analysis in human cells\",\n      \"pmids\": [\"21461783\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interactions shown by co-IP without reciprocal in vitro reconstitution\", \"Functional contribution of HMGA1a-directed targeting not quantified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Used acute cell-cycle-stage-specific depletion to prove the cytokinesis/abscission role is direct and resides in the C-terminal 25 residues, separating it from replication-stress phenotypes.\",\n      \"evidence\": \"N-end rule degron, FRAP-based abscission assay and C-terminal deletion rescue in avian cells\",\n      \"pmids\": [\"21422227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular effectors at the midbody not fully defined\", \"Did not connect to septin biochemistry directly in this system\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated the biochemical consequence of the Orc6-septin interaction—stimulating septin GTPase activity and filament assembly.\",\n      \"evidence\": \"Septin purification, recombinant reconstitution, GTPase assay and electron microscopy of filaments\",\n      \"pmids\": [\"18987337\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of GTPase stimulation during cytokinesis not directly tested\", \"Did not resolve binding stoichiometry\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed Orc6 in the metazoan ORC architecture and tied a Meier-Gorlin syndrome mutation to defective Orc6-Orc3 assembly and MCM loading.\",\n      \"evidence\": \"3D electron microscopy, biochemical binding, in vivo MCM loading and mutagenesis\",\n      \"pmids\": [\"24137536\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the Orc6-Orc3 interface not obtained\", \"Did not survey other disease alleles\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Refined the cytokinesis mechanism by showing Orc6 dimerizes via TFIIB-like domains and bridges septins to promote filament formation.\",\n      \"evidence\": \"Recombinant septin reconstitution, in vitro filament assay and GTP-binding-domain mutagenesis in Drosophila\",\n      \"pmids\": [\"25355953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not determine the bridging structure at atomic resolution\", \"Connection between dimerization and replication function unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Determined the full-length human Orc6 three-domain solution structure and localized a discrete DNA-binding domain confirming its origin-binding role.\",\n      \"evidence\": \"Solution NMR, mutagenesis and in vitro/cell-based replication assays\",\n      \"pmids\": [\"32986843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DNA-bound conformation not captured\", \"Domain interplay within intact ORC not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established a developmental requirement for translational activation of maternal Orc6 mRNA for the first embryonic replication.\",\n      \"evidence\": \"CPE-targeted RNAi maternal mRNA ablation, Western blot and embryo replication assay in mouse\",\n      \"pmids\": [\"20219456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Translational control machinery only inferred from the CPE element\", \"Single-system functional readout\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Uncovered a non-replication chromatin role: Orc6 at the replication fork acts as an accessory factor for mismatch repair by binding MutSα and promoting MutLα loading.\",\n      \"evidence\": \"Co-IP, chromatin fractionation, fork localization and MMR/checkpoint assays in human cells\",\n      \"pmids\": [\"35622890\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Orc6-MutSα binding undefined\", \"Whether this is separable from origin licensing not fully resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified ATR-dependent Thr229 phosphorylation of Orc6 as the trigger for oxidative-damage checkpoint signaling and tumor suppression.\",\n      \"evidence\": \"Phospho-specific antibody, T229A mutagenesis, ATR signaling and tumorigenicity assays\",\n      \"pmids\": [\"37096556\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ATR-Orc6 kinase relationship inferred from dependency\", \"Downstream effectors of phospho-Orc6 not fully mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a CDK-dependent Thr195 phosphorylation that restrains re-licensing outside G1 and linked Orc6 to nucleolar function and ribosome biogenesis.\",\n      \"evidence\": \"T195E phosphomimetic mutagenesis, co-IP, cell cycle analysis and ribosome biogenesis assays\",\n      \"pmids\": [\"38867464\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphomimetic does not fully recapitulate endogenous phosphorylation\", \"Mechanism connecting Orc6 to ribosome biogenesis unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Implicated Orc6 in innate immune signaling through a p65 interaction required for LPS-induced NF\\u03baB activation.\",\n      \"evidence\": \"Co-IP, CRISPR knockout, shRNA silencing, cytokine and NF\\u03baB reporter assays with p65-epistasis in macrophages\",\n      \"pmids\": [\"39143485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus indirect Orc6-p65 binding not structurally defined\", \"Relationship to Orc6 replication function unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved how Orc6 enforces double-hexamer assembly by tethering ORC to Mcm2 during the binding-site switch, and how CDK phosphorylation disrupts this to inhibit loading.\",\n      \"evidence\": \"Single-molecule FRET, Orc6 linker mutagenesis, in vitro MCM loading and CDK phosphorylation assays\",\n      \"pmids\": [\"41055997\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise CDK target residues on ORC controlling tethering not all defined\", \"In vivo confirmation of the tethering intermediate pending\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed proteasome-dependent removal of Orc6 from chromatin at S-phase entry is a safeguard against MCM reloading and tetraploidy.\",\n      \"evidence\": \"Chromatin fractionation, proteasome inhibition, MCM loading and ploidy analysis in human cells\",\n      \"pmids\": [\"40554748\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin ligase mediating Orc6 turnover not identified\", \"Direct evidence Orc6 is the proteasome substrate versus indirect effect not fully separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Orc6's distinct activities—origin DNA binding, helicase loading, septin/cytokinesis, mismatch repair, checkpoint signaling, nucleolar/ribosome biogenesis and NF\\u03baB signaling—are spatially and temporally coordinated within a single small protein remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating replication and non-replication functions\", \"Regulatory hierarchy among the multiple phosphorylation events undefined\", \"Structure of Orc6 bound simultaneously to DNA and partner proteins lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6, 10, 16]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [8, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [6, 21]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [7, 10, 20]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 12, 19]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [17, 18]}\n    ],\n    \"complexes\": [\"origin recognition complex (ORC)\", \"septin complex\"],\n    \"partners\": [\"CDT1\", \"MCM2\", \"ORC3\", \"CDC6\", \"Pnut\", \"MutSα\", \"RELA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}