{"gene":"ORC2","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1996,"finding":"Xenopus Orc2-related protein (XORC2) is required for chromosomal DNA replication: immunodepletion of XORC2 from Xenopus egg extracts abolishes replication of chromosomal DNA but not elongation synthesis on single-stranded DNA templates. XORC2 binds chromatin prior to DNA synthesis and prior to loading of replication licensing factors.","method":"Immunodepletion from Xenopus egg extracts; indirect immunofluorescence","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunodepletion functional assay in cell-free system, replicated conceptually across multiple labs and organisms","pmids":["8552193"],"is_preprint":false},{"year":1996,"finding":"Fission yeast Orp2 (ORC2 ortholog) physically interacts with Cdc2 kinase and with the replication activator Cdc18, and is required for DNA replication; cells lacking Orp2 undergo aberrant mitosis indicating Orp2 is involved in generating a checkpoint signal.","method":"Genetic interaction screen, co-immunoprecipitation, loss-of-function analysis in S. pombe","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical data in a clean yeast system, published simultaneously with the Xenopus paper","pmids":["8552194"],"is_preprint":false},{"year":2000,"finding":"Drosophila ORC2 is required for normal replication timing: two alleles of Drosophila ORC2 disrupt the normal early-euchromatin/late-heterochromatin replication pattern, causing some euchromatic regions to replicate abnormally late, and resulting in defective mitotic chromosome condensation in those late-replicating regions.","method":"Genetic mutant analysis, BrdU incorporation timing assay, cytological examination of mitotic chromosomes in Drosophila larvae","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent alleles with consistent phenotypes, multiple orthogonal readouts (replication timing and condensation)","pmids":["11137005"],"is_preprint":false},{"year":2004,"finding":"Human ORC2 localizes to centrosomes throughout the entire cell cycle and to centromeres and heterochromatin in a cell-cycle-dependent manner. ORC2 is tightly bound to heterochromatin and HP1α/HP1β during G1 and early S phase, but during late S, G2, and M phases chromatin association is restricted to centromeres. Depletion of ORC2 by siRNA disrupts HP1 localization (without affecting H3K9 methylation), causes S-phase defects with reduced PCNA on chromatin (though MCM proteins remain), induces abnormal chromosome condensation, failed chromosome congression, and multiple centrosomes.","method":"siRNA depletion, immunofluorescence, cell fractionation, co-immunoprecipitation with HP1α and HP1β","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (siRNA, IF, fractionation, Co-IP), multiple distinct phenotypic readouts in a single rigorous study","pmids":["15215892"],"is_preprint":false},{"year":2006,"finding":"Human ORC2 contains a single ORC assembly domain required in vivo for interaction with all other ORC subunits, and two nuclear localization signals (NLS) required for ORC accumulation in the nucleus. In the nucleus, ORC2 exists as ORC(2-5) or ORC(1-5) complexes; only ORC(1-5) is chromatin-bound, indicating Orc1 is required to load ORC(2-5) onto chromatin. Additionally, ORC2 suppresses expression of endogenous ORC2, indicating cells limit intracellular ORC2 levels.","method":"Stable expression of epitope-tagged domain-deletion mutants in HeLa cells, chromatin fractionation, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain-mapping mutagenesis with functional fractionation assays, single lab but multiple orthogonal approaches","pmids":["16762929"],"is_preprint":false},{"year":2006,"finding":"Co-expression of ORC2 with ORC1 prevents ORC1-induced apoptosis and restores uniform nuclear localization of ORC1. In the absence of ORC2 co-expression, unmodified ORC1 accumulates perinuclearly and rapidly induces p53-independent apoptosis.","method":"Transient expression of ORC1 ± ORC2 in cell lines, immunofluorescence, apoptosis assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional co-expression rescue experiment, single lab, multiple readouts but no biochemical reconstitution","pmids":["16537645"],"is_preprint":false},{"year":2011,"finding":"Polo-like kinase 1 (Plk1) phosphorylates ORC2 at Ser188 in vitro and in vivo. This phosphorylation is enhanced under DNA replication stress (UV, hydroxyurea, gemcitabine, aphidicolin). Cells expressing the unphosphorylatable S188A ORC2 mutant have defective DNA synthesis under stress, fail to maintain functional pre-replicative complex, and activate the intra-S-phase checkpoint.","method":"In vitro kinase assay, in vivo phosphorylation (mass spectrometry), site-directed mutagenesis (S188A), BrdU incorporation, pre-RC chromatin binding assays, checkpoint activation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus in vivo validation, single lab but multiple orthogonal methods","pmids":["21947279"],"is_preprint":false},{"year":2012,"finding":"Cyclin-dependent kinase phosphorylates ORC2 at Thr-116 and Thr-226 during S phase, causing dissociation of ORC2, ORC3, ORC4, and ORC5 subunits from chromatin and replication origins. Phosphomimetic ORC2 shows defective binding to replication origins and chromatin; phosphodefective ORC2 persists in chromatin binding throughout the cell cycle.","method":"In vitro CDK phosphorylation assay, phosphomimetic and phosphodefective mutagenesis, chromatin fractionation, ChIP at replication origins","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, mutagenesis, and chromatin binding assays in a single study with multiple orthogonal approaches","pmids":["22334659"],"is_preprint":false},{"year":2012,"finding":"ORC2 protects the ORC-associated protein ORCA/LRWD1 from ubiquitin-mediated degradation. ORCA is polyubiquitinated via K48-linked chains by Cul4A-DDB1 E3 ligase at the WD40 repeat domain. ORC2 binds exclusively the non-ubiquitinated form of ORCA, and depletion of ORC2 leads to proteasome-mediated destabilization of ORCA.","method":"In vivo ubiquitination assay, co-immunoprecipitation, siRNA knockdown, proteasome inhibitor treatment","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional knockdown with defined proteasomal readout, single lab","pmids":["22935713"],"is_preprint":false},{"year":2012,"finding":"Plk1 phosphorylation of ORC2 (at Ser188) maintains DNA replication under gemcitabine treatment; cells expressing a Plk1-unphosphorylatable ORC2 mutant are more sensitive to gemcitabine than wild-type ORC2-expressing cells.","method":"Phosphomimetic/phosphodefective ORC2 mutant expression in cancer cells, drug sensitivity assays, mouse xenograft model","journal":"Molecular cancer therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis validated in cell lines and in vivo xenograft, single lab, extends prior mechanistic findings","pmids":["23188630"],"is_preprint":false},{"year":2014,"finding":"Protein phosphatase 1 (PP1) physically interacts with ORC2 via the consensus PP1-binding motif 119-KSVSF-123 on ORC2. PP1 dephosphorylates ORC2 at Thr116 and Thr226 in a cell-cycle-dependent manner (late M phase), which is required for re-binding of ORC2 and associated subunits to chromatin and replication origins.","method":"Co-immunoprecipitation of PP1 and ORC2, PP1 inhibitor treatment, overexpression and siRNA knockdown of PP1 isoforms, chromatin fractionation","journal":"Biochemical and biophysical research communications / Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction demonstrated by Co-IP, dephosphorylation confirmed by PP1 isoform manipulation, single lab across two papers","pmids":["24732362","24792176"],"is_preprint":false},{"year":2016,"finding":"ORC2 is SUMOylated by SUMO2 (but not SUMO1) at the G2/M phase of the cell cycle. SUMO2-modified ORC2 recruits the histone demethylase KDM5A to centromeres to convert H3K4me3 to H3K4me2, a permissive histone mark for α-satellite transcription. Loss of ORC2 SUMOylation (SUMO-less ORC2) reduces α-satellite transcription, impairs pericentric heterochromatin silencing, leads to heterochromatin DNA re-replication, activates DNA damage response, and causes polyploidy.","method":"In vivo SUMOylation assays, co-immunoprecipitation of KDM5A with SUMO2-ORC2, stable expression of SUMO-less ORC2 mutant, chromatin analysis, flow cytometry","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — SUMOylation site mapping, KDM5A recruitment validated by Co-IP, rescue experiments with SUMO-less mutant, multiple orthogonal functional readouts","pmids":["27052177"],"is_preprint":false},{"year":2016,"finding":"Papillomavirus E2 protein binds ORC2; however, ORC2 is not detected at the viral origin. ORC2 depletion enhances PV replication and increases E1/E2 occupancy at the viral origin, indicating ORC2 suppresses E2 replicative function rather than promoting viral replication. Over-expression of HPV E2 decreases ORC2 occupation at mammalian replication origins.","method":"Co-immunoprecipitation of E2 and ORC2, siRNA depletion of ORC2, transient replication assay, ChIP at viral and cellular origins","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional depletion with ChIP validation, single lab","pmids":["27701460"],"is_preprint":false},{"year":2017,"finding":"ORC2 SUMOylation is reversibly regulated: SUMO E3 ligase PIAS4 promotes ORC2 SUMOylation at G2/M, while de-SUMOylase SENP2 removes it. Depletion of PIAS4 or overexpression of SENP2 eliminates ORC2 SUMOylation, causes abnormal centromeric H3K4 methylation, and results in mitotic bypass and polyploidy; co-expression of ORC2-SUMO2 fusion protein reduces polyploid cell formation.","method":"PIAS4 siRNA depletion, SENP2 overexpression, ORC2-SUMO2 fusion rescue, flow cytometry, histone methylation analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SUMO writer/eraser identification with rescue by SUMO fusion, single lab, extends prior SUMOylation findings","pmids":["29050267"],"is_preprint":false},{"year":2019,"finding":"The ORC2 winged-helix domain (WHD) binds dsDNA through a flexible β-sheet hairpin anchor region with key residues R540, K548, and K549. NMR chemical shift perturbations reveal a unique dsDNA binding topology distinct from archaeal and yeast ORC WHDs; mutagenesis of these residues validates their importance for binding.","method":"Crystal structure determination, NMR backbone assignments and chemical shift perturbation, site-directed mutagenesis of DNA-binding residues, molecular dynamics simulation","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus NMR plus mutagenesis validation, single lab but multiple orthogonal structural/biochemical methods","pmids":["30963726"],"is_preprint":false},{"year":2020,"finding":"Human HCT116 cancer cells can survive and maintain normal MCM2-7 chromatin loading and origin firing even when both ORC2 and ORC5 proteins are eliminated by CRISPR-Cas9 mutation, causing destabilization of ORC1, ORC3, and ORC4 as well. This demonstrates that in these selected cancer cells, the six-subunit ORC is not strictly required for MCM2-7 loading or origin specification.","method":"CRISPR-Cas9 gene editing, chromatin fractionation, origin firing assays, Western blot","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic knockouts with multiple functional readouts, single lab but important negative/dispensability finding","pmids":["32989049"],"is_preprint":false},{"year":2024,"finding":"The ORFIUS complex (BRD1, HBO1, BRCA1, BARD1) promotes ORC2 localization at replication origins. Depletion of BRD1 and/or HBO1 reduces origin firing and reduces the number of nuclei with ORC2 foci. BRCA1 regulates BRD1, HBO1, and ORC2 localization at origins; in BRCA1-mutant HGSC cells, ORC2 remains at origins and is unresponsive to replication stress signals.","method":"siRNA/CRISPR depletion of complex components, immunofluorescence for ORC2 foci, origin firing assays, ChIP","journal":"NAR cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complex depletion with multiple functional readouts including ChIP, single lab","pmids":["38288445"],"is_preprint":false},{"year":2025,"finding":"DNA-bound ORC2 (independent of the full six-subunit ORC) compacts chromatin and attracts repressive histone marks at focal genomic sites, while also activating chromatin and protecting genes from repressive marks at other sites. ORC2 also prevents CTCF acquisition at focal sites to regulate chromatin loops and indirectly affects epigenetics. Individual ORC subunits bind thousands of sites without co-occupancy of other subunits.","method":"Multi-omics analysis (ChIP-seq, ATAC-seq, Hi-C) in ORC-mutant cancer cell lines (ORC1, ORC2, ORC5 CRISPR knockouts)","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omics in genetically defined cell lines, single lab, genome-wide functional data but no in vitro reconstitution","pmids":["40504688"],"is_preprint":false},{"year":2002,"finding":"Elevated cyclin A-dependent kinase activity in Xenopus egg extract prevents XORC2 from binding to chromatin from permeable erythrocyte nuclei; kinase inhibition reverses this effect. However, inhibiting nuclear-accumulated kinase activity within intact erythrocyte nuclei does not facilitate XORC2 binding to chromatin, suggesting additional mechanisms prevent ORC association within intact terminally differentiated nuclei.","method":"Xenopus egg extract replication system, permeable and intact erythrocyte nuclei, cyclin A-CDK titration, kinase inhibitor treatment","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — cell-free system with kinase manipulation, single lab, partially negative finding for intact nuclei","pmids":["11900493"],"is_preprint":false},{"year":2011,"finding":"Sp1 transcription factor participates in recruiting ORC2 to the chicken lysozyme GAS41 replication origin; knockdown of Sp1 by RNA interference reduces specific ORC2 binding to this origin, which maps to a region containing multiple Sp1/Sp3-binding sites coinciding with DNase I hypersensitive sites.","method":"Chromatin immunoprecipitation (ChIP), quantitative real-time PCR, siRNA knockdown of Sp1","journal":"DNA and cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single ChIP-based study, indirect evidence via Sp1 knockdown, single lab","pmids":["21879882"],"is_preprint":false}],"current_model":"ORC2 is a conserved subunit of the origin recognition complex that is required for chromosomal DNA replication initiation; it is phosphorylated by cyclin A/CDK2 (at T116 and T226) to dissociate ORC from chromatin during S phase and re-loaded by PP1-mediated dephosphorylation in late M phase, phosphorylated by Plk1 (at S188) to maintain replication under stress, and SUMOylated by PIAS4 (reversibly removed by SENP2) at G2/M to recruit the histone demethylase KDM5A to centromeres to regulate centromeric histone methylation and prevent DNA re-replication; beyond replication, DNA-bound ORC2 independently regulates chromatin compaction, repressive histone marks, CTCF binding, and chromosome loop structure at thousands of genomic sites."},"narrative":{"mechanistic_narrative":"ORC2 is a conserved subunit of the origin recognition complex that is essential for the initiation of chromosomal DNA replication, binding chromatin prior to DNA synthesis and prior to loading of replication licensing factors [PMID:8552193, PMID:8552194]. Within human ORC, ORC2 provides an assembly platform: a single ORC assembly domain mediates interaction with all other ORC subunits and two nuclear localization signals drive nuclear accumulation, with ORC2 existing as ORC(2-5) subcomplexes that require ORC1 to load onto chromatin [PMID:16762929], and co-expression with ORC1 suppresses ORC1-induced apoptosis [PMID:16537645]. Its winged-helix domain binds double-stranded DNA through a flexible β-hairpin anchor (R540, K548, K549) using a topology distinct from archaeal and yeast ORC [PMID:30963726]. ORC2 chromatin association is dynamically controlled across the cell cycle by post-translational modification: cyclin/CDK phosphorylation at Thr116 and Thr226 dissociates ORC2–5 from origins during S phase, and PP1, recruited via the KSVSF motif on ORC2, reverses this in late M phase to permit re-loading [PMID:22334659, PMID:24732362, PMID:24792176, PMID:11900493]; Plk1 phosphorylation at Ser188 sustains replication and the pre-replicative complex under replication stress [PMID:21947279, PMID:23188630]. At G2/M, PIAS4-mediated SUMOylation by SUMO2 (reversed by SENP2) recruits the histone demethylase KDM5A to centromeres to convert H3K4me3 to H3K4me2, supporting α-satellite transcription, pericentric heterochromatin silencing, and prevention of DNA re-replication [PMID:27052177, PMID:29050267]. ORC2 localizes to centrosomes, centromeres, and heterochromatin and associates with HP1, with its loss causing chromosome condensation, congression, and centrosome defects [PMID:15215892], and it regulates replication timing of euchromatin versus heterochromatin [PMID:11137005]. Beyond canonical licensing, DNA-bound ORC2 independent of the full six-subunit complex compacts chromatin, attracts or excludes repressive histone marks, and restricts CTCF binding to shape chromosome loops at thousands of genomic sites [PMID:40504688]; notably, selected cancer cells survive complete ORC2/ORC5 loss with intact MCM2-7 loading, indicating the intact six-subunit ORC is not strictly required for origin firing in this context [PMID:32989049].","teleology":[{"year":1996,"claim":"Established that ORC2 is required for the initiation step of chromosomal DNA replication and acts upstream of licensing, defining its core biological role.","evidence":"Immunodepletion of XORC2 from Xenopus egg extracts and chromatin-binding assays; reciprocal genetic and Co-IP analysis of fission yeast Orp2 with Cdc2 and Cdc18","pmids":["8552193","8552194"],"confidence":"High","gaps":["Did not resolve the molecular mechanism of origin selection","Checkpoint signaling role inferred from loss-of-function but not mechanistically dissected"]},{"year":2000,"claim":"Showed that ORC2 controls genome-wide replication timing, coupling origin function to chromatin state and mitotic condensation.","evidence":"Genetic mutant analysis with BrdU timing and cytology of mitotic chromosomes in Drosophila larvae","pmids":["11137005"],"confidence":"High","gaps":["Mechanism linking ORC2 to late-replication delay not defined","Connection between replication timing and condensation defect correlative"]},{"year":2004,"claim":"Defined the cell-cycle-dependent localization of human ORC2 to centrosomes, centromeres, and heterochromatin and its physical association with HP1, expanding its role beyond replication into chromosome architecture.","evidence":"siRNA depletion, immunofluorescence, cell fractionation, and Co-IP with HP1α/HP1β in human cells","pmids":["15215892"],"confidence":"High","gaps":["Whether HP1 association is direct not established","Mechanistic basis of centrosome localization unknown"]},{"year":2006,"claim":"Mapped the ORC2 assembly domain and NLS signals and showed ORC1-dependence of chromatin loading, clarifying how ORC2 nucleates and positions the complex.","evidence":"Domain-deletion mutant expression, chromatin fractionation, and Co-IP in HeLa cells; ORC1/ORC2 co-expression apoptosis rescue assays","pmids":["16762929","16537645"],"confidence":"High","gaps":["Structural basis of subunit contacts not resolved","ORC1-induced apoptosis pathway only phenomenologically described"]},{"year":2012,"claim":"Identified CDK phosphorylation at Thr116/Thr226 as the switch that dissociates ORC2 and partner subunits from origins during S phase, providing a mechanism for replication licensing control.","evidence":"In vitro CDK kinase assay, phosphomimetic/phosphodefective mutagenesis, chromatin fractionation, and origin ChIP","pmids":["22334659"],"confidence":"High","gaps":["Did not identify the re-loading mechanism (resolved later by PP1 work)"]},{"year":2014,"claim":"Showed PP1 directly binds ORC2 via the KSVSF motif and dephosphorylates Thr116/Thr226 in late M phase, closing the loop on cyclic ORC2 chromatin re-loading.","evidence":"Co-IP of PP1 with ORC2, PP1 inhibitor and isoform manipulation, chromatin fractionation","pmids":["24732362","24792176"],"confidence":"Medium","gaps":["PP1 isoform specificity in vivo not fully resolved","Single lab across two papers"]},{"year":2012,"claim":"Established a stress-responsive arm in which Plk1 phosphorylates ORC2 at Ser188 to maintain the pre-RC and replication under genotoxic stress.","evidence":"In vitro kinase assay, mass-spec phosphosite mapping, S188A mutagenesis, BrdU and checkpoint assays; drug sensitivity and xenograft validation","pmids":["21947279","23188630"],"confidence":"High","gaps":["Downstream effectors of S188 phosphorylation not defined","How Plk1 and CDK phosphorylation are coordinated unclear"]},{"year":2012,"claim":"Connected ORC2 to stability of the ORC-associated factor ORCA/LRWD1, showing ORC2 binds and protects the non-ubiquitinated form from proteasomal degradation.","evidence":"In vivo ubiquitination assay, reciprocal Co-IP, siRNA knockdown, proteasome inhibition","pmids":["22935713"],"confidence":"Medium","gaps":["Whether protection is direct steric blocking not shown","Single lab"]},{"year":2016,"claim":"Revealed a SUMO2-dependent function of ORC2 at G2/M that recruits KDM5A to centromeres, linking ORC2 modification to centromeric histone methylation, α-satellite transcription, and re-replication suppression.","evidence":"In vivo SUMOylation site mapping, Co-IP of KDM5A with SUMO2-ORC2, SUMO-less rescue, chromatin analysis and flow cytometry","pmids":["27052177"],"confidence":"High","gaps":["Direct SUMO-KDM5A interaction interface not defined","Whether this is replication-independent not fully separated"]},{"year":2017,"claim":"Identified the writer/eraser pair (PIAS4/SENP2) governing reversible ORC2 SUMOylation, establishing the enzymatic control of the centromeric methylation circuit.","evidence":"PIAS4 knockdown, SENP2 overexpression, ORC2-SUMO2 fusion rescue, flow cytometry and histone methylation analysis","pmids":["29050267"],"confidence":"Medium","gaps":["Upstream signals triggering G2/M SUMOylation unknown","Single lab extending prior work"]},{"year":2019,"claim":"Provided the structural basis for ORC2 DNA engagement, defining a winged-helix domain with a distinct dsDNA-binding topology.","evidence":"Crystal structure, NMR chemical shift perturbation, mutagenesis of R540/K548/K549, molecular dynamics","pmids":["30963726"],"confidence":"High","gaps":["Structure of full ORC2 within the assembled complex not resolved","Sequence specificity of DNA binding not addressed"]},{"year":2020,"claim":"Demonstrated that the intact six-subunit ORC, including ORC2, is dispensable for MCM2-7 loading and origin firing in selected cancer cells, challenging the obligatory role of ORC2 in licensing.","evidence":"CRISPR-Cas9 ORC2/ORC5 knockout, chromatin fractionation, origin firing assays, Western blot in HCT116","pmids":["32989049"],"confidence":"Medium","gaps":["How origins are loaded without ORC unresolved","May reflect adaptation specific to these selected cells"]},{"year":2024,"claim":"Placed ORC2 origin localization downstream of the ORFIUS (BRD1/HBO1/BRCA1/BARD1) complex, linking ORC2 recruitment to a chromatin-modifying complex and replication stress responsiveness.","evidence":"siRNA/CRISPR depletion of complex components, ORC2 foci immunofluorescence, origin firing assays, ChIP","pmids":["38288445"],"confidence":"Medium","gaps":["Direct versus indirect recruitment by ORFIUS not separated","Mechanism of stress unresponsiveness in BRCA1-mutant cells unclear"]},{"year":2025,"claim":"Defined a replication-independent genomic role for DNA-bound ORC2 in chromatin compaction, histone mark deposition/protection, and CTCF/loop regulation at thousands of sites.","evidence":"Multi-omics (ChIP-seq, ATAC-seq, Hi-C) in ORC-knockout cancer cell lines","pmids":["40504688"],"confidence":"Medium","gaps":["No in vitro reconstitution of ORC2-driven compaction","Mechanism by which individual subunits bind without co-occupancy unknown"]},{"year":null,"claim":"How the multiple ORC2 modification circuits (CDK, PP1, Plk1, SUMO) are integrated, and how replication-independent chromatin functions relate to the dispensability of intact ORC in some cells, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coordinating the four modification axes","Mechanism of ORC-independent MCM loading unknown","In vitro reconstitution of the chromatin-architecture role lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[14,7,17]}],"localization":[{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[3,7,17]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4,3]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0,7,6]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[11,17,3]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7,10]}],"complexes":["Origin recognition complex (ORC)"],"partners":["ORC1","ORC3","ORC4","ORC5","HP1","KDM5A","PP1","ORCA/LRWD1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BXI2","full_name":"Mitochondrial ornithine transporter 2","aliases":["Solute carrier family 25 member 2"],"length_aa":301,"mass_kda":32.6,"function":"Mitochondrial transporter of the positively charged amino acids ornithine, lysine and arginine, and the neutral amino acid citrulline (PubMed:12807890). In addition, transports the basic amino acids histidine, homoarginine, and asymmetric dimethylarginine (aDMA), but not symmetric DMA, and the D-forms of lysine, arginine, ornithine and histidine (PubMed:12807890, PubMed:26403849). Functions by both counter-exchange and uniport mechanisms (PubMed:26403849)","subcellular_location":"Mitochondrion membrane; Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/Q9BXI2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ORC2","classification":"Not Classified","n_dependent_lines":112,"n_total_lines":1208,"dependency_fraction":0.09271523178807947},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DDX6","stoichiometry":0.2},{"gene":"FXR1","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ORC2","total_profiled":1310},"omim":[{"mim_id":"615167","title":"LEUCINE-RICH REPEATS- AND WD REPEAT DOMAIN-CONTAINING PROTEIN 1; LRWD1","url":"https://www.omim.org/entry/615167"},{"mim_id":"613362","title":"CDK2-INTERACTING PROTEIN; CINP","url":"https://www.omim.org/entry/613362"},{"mim_id":"609357","title":"MINICHROMOSOME MAINTENANCE COMPLEX COMPONENT 10; MCM10","url":"https://www.omim.org/entry/609357"},{"mim_id":"605525","title":"CHROMATIN LICENSING AND DNA REPLICATION FACTOR 1; CDT1","url":"https://www.omim.org/entry/605525"},{"mim_id":"603056","title":"ORIGIN RECOGNITION COMPLEX, SUBUNIT 4; ORC4","url":"https://www.omim.org/entry/603056"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ORC2"},"hgnc":{"alias_symbol":[],"prev_symbol":["ORC2L"]},"alphafold":{"accession":"Q9BXI2","domains":[{"cath_id":"1.50.40.10","chopping":"6-301","consensus_level":"high","plddt":88.8059,"start":6,"end":301}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXI2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXI2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXI2-F1-predicted_aligned_error_v6.png","plddt_mean":88.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ORC2","jax_strain_url":"https://www.jax.org/strain/search?query=ORC2"},"sequence":{"accession":"Q9BXI2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BXI2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BXI2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXI2"}},"corpus_meta":[{"pmid":"15215892","id":"PMC_15215892","title":"Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance.","date":"2004","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/15215892","citation_count":228,"is_preprint":false},{"pmid":"8552193","id":"PMC_8552193","title":"Role for a Xenopus Orc2-related protein in controlling DNA replication.","date":"1996","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8552193","citation_count":185,"is_preprint":false},{"pmid":"8552194","id":"PMC_8552194","title":"Interaction of Cdc2 and Cdc18 with a fission yeast ORC2-like protein.","date":"1996","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8552194","citation_count":123,"is_preprint":false},{"pmid":"11137005","id":"PMC_11137005","title":"Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants.","date":"2000","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/11137005","citation_count":99,"is_preprint":false},{"pmid":"23188630","id":"PMC_23188630","title":"Plk1 phosphorylation of orc2 and hbo1 contributes to gemcitabine resistance in pancreatic cancer.","date":"2012","source":"Molecular cancer therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/23188630","citation_count":70,"is_preprint":false},{"pmid":"21947279","id":"PMC_21947279","title":"Plk1 phosphorylation of Orc2 promotes DNA replication under conditions of stress.","date":"2011","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21947279","citation_count":63,"is_preprint":false},{"pmid":"16537645","id":"PMC_16537645","title":"Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis.","date":"2006","source":"Journal of cell 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analysis of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear localization of the origin recognition complex.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16762929","citation_count":15,"is_preprint":false},{"pmid":"22674395","id":"PMC_22674395","title":"Unique pattern of ORC2 and MCM7 localization during DNA replication licensing in the mouse zygote.","date":"2012","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/22674395","citation_count":10,"is_preprint":false},{"pmid":"20626860","id":"PMC_20626860","title":"Global expression studies in baker's yeast reveal target genes for the improvement of industrially-relevant traits: the cases of CAF16 and ORC2.","date":"2010","source":"Microbial cell factories","url":"https://pubmed.ncbi.nlm.nih.gov/20626860","citation_count":9,"is_preprint":false},{"pmid":"24792176","id":"PMC_24792176","title":"Dephosphorylation of Orc2 by 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XORC2 binds chromatin prior to DNA synthesis and prior to loading of replication licensing factors.\",\n      \"method\": \"Immunodepletion from Xenopus egg extracts; indirect immunofluorescence\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunodepletion functional assay in cell-free system, replicated conceptually across multiple labs and organisms\",\n      \"pmids\": [\"8552193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Fission yeast Orp2 (ORC2 ortholog) physically interacts with Cdc2 kinase and with the replication activator Cdc18, and is required for DNA replication; cells lacking Orp2 undergo aberrant mitosis indicating Orp2 is involved in generating a checkpoint signal.\",\n      \"method\": \"Genetic interaction screen, co-immunoprecipitation, loss-of-function analysis in S. pombe\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical data in a clean yeast system, published simultaneously with the Xenopus paper\",\n      \"pmids\": [\"8552194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Drosophila ORC2 is required for normal replication timing: two alleles of Drosophila ORC2 disrupt the normal early-euchromatin/late-heterochromatin replication pattern, causing some euchromatic regions to replicate abnormally late, and resulting in defective mitotic chromosome condensation in those late-replicating regions.\",\n      \"method\": \"Genetic mutant analysis, BrdU incorporation timing assay, cytological examination of mitotic chromosomes in Drosophila larvae\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent alleles with consistent phenotypes, multiple orthogonal readouts (replication timing and condensation)\",\n      \"pmids\": [\"11137005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human ORC2 localizes to centrosomes throughout the entire cell cycle and to centromeres and heterochromatin in a cell-cycle-dependent manner. ORC2 is tightly bound to heterochromatin and HP1α/HP1β during G1 and early S phase, but during late S, G2, and M phases chromatin association is restricted to centromeres. Depletion of ORC2 by siRNA disrupts HP1 localization (without affecting H3K9 methylation), causes S-phase defects with reduced PCNA on chromatin (though MCM proteins remain), induces abnormal chromosome condensation, failed chromosome congression, and multiple centrosomes.\",\n      \"method\": \"siRNA depletion, immunofluorescence, cell fractionation, co-immunoprecipitation with HP1α and HP1β\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (siRNA, IF, fractionation, Co-IP), multiple distinct phenotypic readouts in a single rigorous study\",\n      \"pmids\": [\"15215892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Human ORC2 contains a single ORC assembly domain required in vivo for interaction with all other ORC subunits, and two nuclear localization signals (NLS) required for ORC accumulation in the nucleus. In the nucleus, ORC2 exists as ORC(2-5) or ORC(1-5) complexes; only ORC(1-5) is chromatin-bound, indicating Orc1 is required to load ORC(2-5) onto chromatin. Additionally, ORC2 suppresses expression of endogenous ORC2, indicating cells limit intracellular ORC2 levels.\",\n      \"method\": \"Stable expression of epitope-tagged domain-deletion mutants in HeLa cells, chromatin fractionation, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-mapping mutagenesis with functional fractionation assays, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"16762929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Co-expression of ORC2 with ORC1 prevents ORC1-induced apoptosis and restores uniform nuclear localization of ORC1. In the absence of ORC2 co-expression, unmodified ORC1 accumulates perinuclearly and rapidly induces p53-independent apoptosis.\",\n      \"method\": \"Transient expression of ORC1 ± ORC2 in cell lines, immunofluorescence, apoptosis assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional co-expression rescue experiment, single lab, multiple readouts but no biochemical reconstitution\",\n      \"pmids\": [\"16537645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Polo-like kinase 1 (Plk1) phosphorylates ORC2 at Ser188 in vitro and in vivo. This phosphorylation is enhanced under DNA replication stress (UV, hydroxyurea, gemcitabine, aphidicolin). Cells expressing the unphosphorylatable S188A ORC2 mutant have defective DNA synthesis under stress, fail to maintain functional pre-replicative complex, and activate the intra-S-phase checkpoint.\",\n      \"method\": \"In vitro kinase assay, in vivo phosphorylation (mass spectrometry), site-directed mutagenesis (S188A), BrdU incorporation, pre-RC chromatin binding assays, checkpoint activation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus in vivo validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21947279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cyclin-dependent kinase phosphorylates ORC2 at Thr-116 and Thr-226 during S phase, causing dissociation of ORC2, ORC3, ORC4, and ORC5 subunits from chromatin and replication origins. Phosphomimetic ORC2 shows defective binding to replication origins and chromatin; phosphodefective ORC2 persists in chromatin binding throughout the cell cycle.\",\n      \"method\": \"In vitro CDK phosphorylation assay, phosphomimetic and phosphodefective mutagenesis, chromatin fractionation, ChIP at replication origins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, mutagenesis, and chromatin binding assays in a single study with multiple orthogonal approaches\",\n      \"pmids\": [\"22334659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ORC2 protects the ORC-associated protein ORCA/LRWD1 from ubiquitin-mediated degradation. ORCA is polyubiquitinated via K48-linked chains by Cul4A-DDB1 E3 ligase at the WD40 repeat domain. ORC2 binds exclusively the non-ubiquitinated form of ORCA, and depletion of ORC2 leads to proteasome-mediated destabilization of ORCA.\",\n      \"method\": \"In vivo ubiquitination assay, co-immunoprecipitation, siRNA knockdown, proteasome inhibitor treatment\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional knockdown with defined proteasomal readout, single lab\",\n      \"pmids\": [\"22935713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Plk1 phosphorylation of ORC2 (at Ser188) maintains DNA replication under gemcitabine treatment; cells expressing a Plk1-unphosphorylatable ORC2 mutant are more sensitive to gemcitabine than wild-type ORC2-expressing cells.\",\n      \"method\": \"Phosphomimetic/phosphodefective ORC2 mutant expression in cancer cells, drug sensitivity assays, mouse xenograft model\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis validated in cell lines and in vivo xenograft, single lab, extends prior mechanistic findings\",\n      \"pmids\": [\"23188630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Protein phosphatase 1 (PP1) physically interacts with ORC2 via the consensus PP1-binding motif 119-KSVSF-123 on ORC2. PP1 dephosphorylates ORC2 at Thr116 and Thr226 in a cell-cycle-dependent manner (late M phase), which is required for re-binding of ORC2 and associated subunits to chromatin and replication origins.\",\n      \"method\": \"Co-immunoprecipitation of PP1 and ORC2, PP1 inhibitor treatment, overexpression and siRNA knockdown of PP1 isoforms, chromatin fractionation\",\n      \"journal\": \"Biochemical and biophysical research communications / Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction demonstrated by Co-IP, dephosphorylation confirmed by PP1 isoform manipulation, single lab across two papers\",\n      \"pmids\": [\"24732362\", \"24792176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ORC2 is SUMOylated by SUMO2 (but not SUMO1) at the G2/M phase of the cell cycle. SUMO2-modified ORC2 recruits the histone demethylase KDM5A to centromeres to convert H3K4me3 to H3K4me2, a permissive histone mark for α-satellite transcription. Loss of ORC2 SUMOylation (SUMO-less ORC2) reduces α-satellite transcription, impairs pericentric heterochromatin silencing, leads to heterochromatin DNA re-replication, activates DNA damage response, and causes polyploidy.\",\n      \"method\": \"In vivo SUMOylation assays, co-immunoprecipitation of KDM5A with SUMO2-ORC2, stable expression of SUMO-less ORC2 mutant, chromatin analysis, flow cytometry\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — SUMOylation site mapping, KDM5A recruitment validated by Co-IP, rescue experiments with SUMO-less mutant, multiple orthogonal functional readouts\",\n      \"pmids\": [\"27052177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Papillomavirus E2 protein binds ORC2; however, ORC2 is not detected at the viral origin. ORC2 depletion enhances PV replication and increases E1/E2 occupancy at the viral origin, indicating ORC2 suppresses E2 replicative function rather than promoting viral replication. Over-expression of HPV E2 decreases ORC2 occupation at mammalian replication origins.\",\n      \"method\": \"Co-immunoprecipitation of E2 and ORC2, siRNA depletion of ORC2, transient replication assay, ChIP at viral and cellular origins\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional depletion with ChIP validation, single lab\",\n      \"pmids\": [\"27701460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ORC2 SUMOylation is reversibly regulated: SUMO E3 ligase PIAS4 promotes ORC2 SUMOylation at G2/M, while de-SUMOylase SENP2 removes it. Depletion of PIAS4 or overexpression of SENP2 eliminates ORC2 SUMOylation, causes abnormal centromeric H3K4 methylation, and results in mitotic bypass and polyploidy; co-expression of ORC2-SUMO2 fusion protein reduces polyploid cell formation.\",\n      \"method\": \"PIAS4 siRNA depletion, SENP2 overexpression, ORC2-SUMO2 fusion rescue, flow cytometry, histone methylation analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SUMO writer/eraser identification with rescue by SUMO fusion, single lab, extends prior SUMOylation findings\",\n      \"pmids\": [\"29050267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The ORC2 winged-helix domain (WHD) binds dsDNA through a flexible β-sheet hairpin anchor region with key residues R540, K548, and K549. NMR chemical shift perturbations reveal a unique dsDNA binding topology distinct from archaeal and yeast ORC WHDs; mutagenesis of these residues validates their importance for binding.\",\n      \"method\": \"Crystal structure determination, NMR backbone assignments and chemical shift perturbation, site-directed mutagenesis of DNA-binding residues, molecular dynamics simulation\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus NMR plus mutagenesis validation, single lab but multiple orthogonal structural/biochemical methods\",\n      \"pmids\": [\"30963726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Human HCT116 cancer cells can survive and maintain normal MCM2-7 chromatin loading and origin firing even when both ORC2 and ORC5 proteins are eliminated by CRISPR-Cas9 mutation, causing destabilization of ORC1, ORC3, and ORC4 as well. This demonstrates that in these selected cancer cells, the six-subunit ORC is not strictly required for MCM2-7 loading or origin specification.\",\n      \"method\": \"CRISPR-Cas9 gene editing, chromatin fractionation, origin firing assays, Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic knockouts with multiple functional readouts, single lab but important negative/dispensability finding\",\n      \"pmids\": [\"32989049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The ORFIUS complex (BRD1, HBO1, BRCA1, BARD1) promotes ORC2 localization at replication origins. Depletion of BRD1 and/or HBO1 reduces origin firing and reduces the number of nuclei with ORC2 foci. BRCA1 regulates BRD1, HBO1, and ORC2 localization at origins; in BRCA1-mutant HGSC cells, ORC2 remains at origins and is unresponsive to replication stress signals.\",\n      \"method\": \"siRNA/CRISPR depletion of complex components, immunofluorescence for ORC2 foci, origin firing assays, ChIP\",\n      \"journal\": \"NAR cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complex depletion with multiple functional readouts including ChIP, single lab\",\n      \"pmids\": [\"38288445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DNA-bound ORC2 (independent of the full six-subunit ORC) compacts chromatin and attracts repressive histone marks at focal genomic sites, while also activating chromatin and protecting genes from repressive marks at other sites. ORC2 also prevents CTCF acquisition at focal sites to regulate chromatin loops and indirectly affects epigenetics. Individual ORC subunits bind thousands of sites without co-occupancy of other subunits.\",\n      \"method\": \"Multi-omics analysis (ChIP-seq, ATAC-seq, Hi-C) in ORC-mutant cancer cell lines (ORC1, ORC2, ORC5 CRISPR knockouts)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omics in genetically defined cell lines, single lab, genome-wide functional data but no in vitro reconstitution\",\n      \"pmids\": [\"40504688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Elevated cyclin A-dependent kinase activity in Xenopus egg extract prevents XORC2 from binding to chromatin from permeable erythrocyte nuclei; kinase inhibition reverses this effect. However, inhibiting nuclear-accumulated kinase activity within intact erythrocyte nuclei does not facilitate XORC2 binding to chromatin, suggesting additional mechanisms prevent ORC association within intact terminally differentiated nuclei.\",\n      \"method\": \"Xenopus egg extract replication system, permeable and intact erythrocyte nuclei, cyclin A-CDK titration, kinase inhibitor treatment\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — cell-free system with kinase manipulation, single lab, partially negative finding for intact nuclei\",\n      \"pmids\": [\"11900493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Sp1 transcription factor participates in recruiting ORC2 to the chicken lysozyme GAS41 replication origin; knockdown of Sp1 by RNA interference reduces specific ORC2 binding to this origin, which maps to a region containing multiple Sp1/Sp3-binding sites coinciding with DNase I hypersensitive sites.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), quantitative real-time PCR, siRNA knockdown of Sp1\",\n      \"journal\": \"DNA and cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single ChIP-based study, indirect evidence via Sp1 knockdown, single lab\",\n      \"pmids\": [\"21879882\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ORC2 is a conserved subunit of the origin recognition complex that is required for chromosomal DNA replication initiation; it is phosphorylated by cyclin A/CDK2 (at T116 and T226) to dissociate ORC from chromatin during S phase and re-loaded by PP1-mediated dephosphorylation in late M phase, phosphorylated by Plk1 (at S188) to maintain replication under stress, and SUMOylated by PIAS4 (reversibly removed by SENP2) at G2/M to recruit the histone demethylase KDM5A to centromeres to regulate centromeric histone methylation and prevent DNA re-replication; beyond replication, DNA-bound ORC2 independently regulates chromatin compaction, repressive histone marks, CTCF binding, and chromosome loop structure at thousands of genomic sites.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ORC2 is a conserved subunit of the origin recognition complex that is essential for the initiation of chromosomal DNA replication, binding chromatin prior to DNA synthesis and prior to loading of replication licensing factors [#0, #1]. Within human ORC, ORC2 provides an assembly platform: a single ORC assembly domain mediates interaction with all other ORC subunits and two nuclear localization signals drive nuclear accumulation, with ORC2 existing as ORC(2-5) subcomplexes that require ORC1 to load onto chromatin [#4], and co-expression with ORC1 suppresses ORC1-induced apoptosis [#5]. Its winged-helix domain binds double-stranded DNA through a flexible β-hairpin anchor (R540, K548, K549) using a topology distinct from archaeal and yeast ORC [#14]. ORC2 chromatin association is dynamically controlled across the cell cycle by post-translational modification: cyclin/CDK phosphorylation at Thr116 and Thr226 dissociates ORC2–5 from origins during S phase, and PP1, recruited via the KSVSF motif on ORC2, reverses this in late M phase to permit re-loading [#7, #10, #18]; Plk1 phosphorylation at Ser188 sustains replication and the pre-replicative complex under replication stress [#6, #9]. At G2/M, PIAS4-mediated SUMOylation by SUMO2 (reversed by SENP2) recruits the histone demethylase KDM5A to centromeres to convert H3K4me3 to H3K4me2, supporting α-satellite transcription, pericentric heterochromatin silencing, and prevention of DNA re-replication [#11, #13]. ORC2 localizes to centrosomes, centromeres, and heterochromatin and associates with HP1, with its loss causing chromosome condensation, congression, and centrosome defects [#3], and it regulates replication timing of euchromatin versus heterochromatin [#2]. Beyond canonical licensing, DNA-bound ORC2 independent of the full six-subunit complex compacts chromatin, attracts or excludes repressive histone marks, and restricts CTCF binding to shape chromosome loops at thousands of genomic sites [#17]; notably, selected cancer cells survive complete ORC2/ORC5 loss with intact MCM2-7 loading, indicating the intact six-subunit ORC is not strictly required for origin firing in this context [#15].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that ORC2 is required for the initiation step of chromosomal DNA replication and acts upstream of licensing, defining its core biological role.\",\n      \"evidence\": \"Immunodepletion of XORC2 from Xenopus egg extracts and chromatin-binding assays; reciprocal genetic and Co-IP analysis of fission yeast Orp2 with Cdc2 and Cdc18\",\n      \"pmids\": [\n        \"8552193\",\n        \"8552194\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not resolve the molecular mechanism of origin selection\",\n        \"Checkpoint signaling role inferred from loss-of-function but not mechanistically dissected\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed that ORC2 controls genome-wide replication timing, coupling origin function to chromatin state and mitotic condensation.\",\n      \"evidence\": \"Genetic mutant analysis with BrdU timing and cytology of mitotic chromosomes in Drosophila larvae\",\n      \"pmids\": [\n        \"11137005\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism linking ORC2 to late-replication delay not defined\",\n        \"Connection between replication timing and condensation defect correlative\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the cell-cycle-dependent localization of human ORC2 to centrosomes, centromeres, and heterochromatin and its physical association with HP1, expanding its role beyond replication into chromosome architecture.\",\n      \"evidence\": \"siRNA depletion, immunofluorescence, cell fractionation, and Co-IP with HP1α/HP1β in human cells\",\n      \"pmids\": [\n        \"15215892\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether HP1 association is direct not established\",\n        \"Mechanistic basis of centrosome localization unknown\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mapped the ORC2 assembly domain and NLS signals and showed ORC1-dependence of chromatin loading, clarifying how ORC2 nucleates and positions the complex.\",\n      \"evidence\": \"Domain-deletion mutant expression, chromatin fractionation, and Co-IP in HeLa cells; ORC1/ORC2 co-expression apoptosis rescue assays\",\n      \"pmids\": [\n        \"16762929\",\n        \"16537645\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of subunit contacts not resolved\",\n        \"ORC1-induced apoptosis pathway only phenomenologically described\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified CDK phosphorylation at Thr116/Thr226 as the switch that dissociates ORC2 and partner subunits from origins during S phase, providing a mechanism for replication licensing control.\",\n      \"evidence\": \"In vitro CDK kinase assay, phosphomimetic/phosphodefective mutagenesis, chromatin fractionation, and origin ChIP\",\n      \"pmids\": [\n        \"22334659\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not identify the re-loading mechanism (resolved later by PP1 work)\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed PP1 directly binds ORC2 via the KSVSF motif and dephosphorylates Thr116/Thr226 in late M phase, closing the loop on cyclic ORC2 chromatin re-loading.\",\n      \"evidence\": \"Co-IP of PP1 with ORC2, PP1 inhibitor and isoform manipulation, chromatin fractionation\",\n      \"pmids\": [\n        \"24732362\",\n        \"24792176\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"PP1 isoform specificity in vivo not fully resolved\",\n        \"Single lab across two papers\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Established a stress-responsive arm in which Plk1 phosphorylates ORC2 at Ser188 to maintain the pre-RC and replication under genotoxic stress.\",\n      \"evidence\": \"In vitro kinase assay, mass-spec phosphosite mapping, S188A mutagenesis, BrdU and checkpoint assays; drug sensitivity and xenograft validation\",\n      \"pmids\": [\n        \"21947279\",\n        \"23188630\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Downstream effectors of S188 phosphorylation not defined\",\n        \"How Plk1 and CDK phosphorylation are coordinated unclear\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected ORC2 to stability of the ORC-associated factor ORCA/LRWD1, showing ORC2 binds and protects the non-ubiquitinated form from proteasomal degradation.\",\n      \"evidence\": \"In vivo ubiquitination assay, reciprocal Co-IP, siRNA knockdown, proteasome inhibition\",\n      \"pmids\": [\n        \"22935713\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether protection is direct steric blocking not shown\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed a SUMO2-dependent function of ORC2 at G2/M that recruits KDM5A to centromeres, linking ORC2 modification to centromeric histone methylation, α-satellite transcription, and re-replication suppression.\",\n      \"evidence\": \"In vivo SUMOylation site mapping, Co-IP of KDM5A with SUMO2-ORC2, SUMO-less rescue, chromatin analysis and flow cytometry\",\n      \"pmids\": [\n        \"27052177\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct SUMO-KDM5A interaction interface not defined\",\n        \"Whether this is replication-independent not fully separated\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified the writer/eraser pair (PIAS4/SENP2) governing reversible ORC2 SUMOylation, establishing the enzymatic control of the centromeric methylation circuit.\",\n      \"evidence\": \"PIAS4 knockdown, SENP2 overexpression, ORC2-SUMO2 fusion rescue, flow cytometry and histone methylation analysis\",\n      \"pmids\": [\n        \"29050267\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Upstream signals triggering G2/M SUMOylation unknown\",\n        \"Single lab extending prior work\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided the structural basis for ORC2 DNA engagement, defining a winged-helix domain with a distinct dsDNA-binding topology.\",\n      \"evidence\": \"Crystal structure, NMR chemical shift perturbation, mutagenesis of R540/K548/K549, molecular dynamics\",\n      \"pmids\": [\n        \"30963726\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structure of full ORC2 within the assembled complex not resolved\",\n        \"Sequence specificity of DNA binding not addressed\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated that the intact six-subunit ORC, including ORC2, is dispensable for MCM2-7 loading and origin firing in selected cancer cells, challenging the obligatory role of ORC2 in licensing.\",\n      \"evidence\": \"CRISPR-Cas9 ORC2/ORC5 knockout, chromatin fractionation, origin firing assays, Western blot in HCT116\",\n      \"pmids\": [\n        \"32989049\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"How origins are loaded without ORC unresolved\",\n        \"May reflect adaptation specific to these selected cells\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed ORC2 origin localization downstream of the ORFIUS (BRD1/HBO1/BRCA1/BARD1) complex, linking ORC2 recruitment to a chromatin-modifying complex and replication stress responsiveness.\",\n      \"evidence\": \"siRNA/CRISPR depletion of complex components, ORC2 foci immunofluorescence, origin firing assays, ChIP\",\n      \"pmids\": [\n        \"38288445\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct versus indirect recruitment by ORFIUS not separated\",\n        \"Mechanism of stress unresponsiveness in BRCA1-mutant cells unclear\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a replication-independent genomic role for DNA-bound ORC2 in chromatin compaction, histone mark deposition/protection, and CTCF/loop regulation at thousands of sites.\",\n      \"evidence\": \"Multi-omics (ChIP-seq, ATAC-seq, Hi-C) in ORC-knockout cancer cell lines\",\n      \"pmids\": [\n        \"40504688\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No in vitro reconstitution of ORC2-driven compaction\",\n        \"Mechanism by which individual subunits bind without co-occupancy unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple ORC2 modification circuits (CDK, PP1, Plk1, SUMO) are integrated, and how replication-independent chromatin functions relate to the dispensability of intact ORC in some cells, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No unified model coordinating the four modification axes\",\n        \"Mechanism of ORC-independent MCM loading unknown\",\n        \"In vitro reconstitution of the chromatin-architecture role lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\n        \"term_id\": \"GO:0003677\",\n        \"supporting_discovery_ids\": [\n          14,\n          7,\n          17\n        ]\n      }\n    ],\n    \"localization\": [\n      {\n        \"term_id\": \"GO:0000228\",\n        \"supporting_discovery_ids\": [\n          3,\n          7,\n          17\n        ]\n      },\n      {\n        \"term_id\": \"GO:0005815\",\n        \"supporting_discovery_ids\": [\n          3\n        ]\n      },\n      {\n        \"term_id\": \"GO:0005634\",\n        \"supporting_discovery_ids\": [\n          4,\n          3\n        ]\n      }\n    ],\n    \"pathway\": [\n      {\n        \"term_id\": \"R-HSA-69306\",\n        \"supporting_discovery_ids\": [\n          0,\n          7,\n          6\n        ]\n      },\n      {\n        \"term_id\": \"R-HSA-4839726\",\n        \"supporting_discovery_ids\": [\n          11,\n          17,\n          3\n        ]\n      },\n      {\n        \"term_id\": \"R-HSA-1640170\",\n        \"supporting_discovery_ids\": [\n          7,\n          10\n        ]\n      }\n    ],\n    \"complexes\": [\n      \"Origin recognition complex (ORC)\"\n    ],\n    \"partners\": [\n      \"ORC1\",\n      \"ORC3\",\n      \"ORC4\",\n      \"ORC5\",\n      \"HP1\",\n      \"KDM5A\",\n      \"PP1\",\n      \"ORCA/LRWD1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}