{"gene":"DIAPH2","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2009,"finding":"The TPR domain at the amino terminus of yeast Dia2 tethers SCF(Dia2) to the replisome progression complex (RPC) by interacting with the RPC components Mrc1 and Ctf4, increasing the local concentration of the E3 ligase at DNA replication forks.","method":"Co-immunoprecipitation, two-hybrid interaction, domain deletion analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with domain mapping, replicated independently in two concurrent papers (PMID 19913425 and PMID 19910927)","pmids":["19913425"],"is_preprint":false},{"year":2009,"finding":"SCF(Dia2) ubiquitinates replisome components Mrc1 and Ctf4 both in vivo and in vitro, and the leucine-rich repeat (LRR) motif of Dia2 is required for regulation of replisome progression while the TPR motif mediates interaction with replisome components and Dia2 stability.","method":"In vitro ubiquitination assay, in vivo ubiquitination, domain deletion analysis, yeast two-hybrid","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination assay plus domain mutagenesis, replicated in companion paper","pmids":["19910927"],"is_preprint":false},{"year":2009,"finding":"Dia2 localizes to replication forks and regulates replication fork progression under hydroxyurea treatment, as shown by ChIP-on-chip analysis.","method":"ChIP-on-chip","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide ChIP with functional context, single lab","pmids":["19910927"],"is_preprint":false},{"year":2015,"finding":"SCF(Dia2) drives ubiquitylation of the Mcm7 subunit of the CMG helicase at the end of replication, and tethering via the TPR domain to the replisome progression complex increases the efficiency of this ubiquitylation both in vitro and in vivo, promoting CMG disassembly through a pathway requiring the Cdc48 segregase. SCF(Dia2) does NOT mediate replisome-specific degradation of Mrc1 and Ctf4.","method":"In vitro ubiquitylation assay, in vivo ubiquitylation, genetic epistasis (synthetic lethality with cdc48 allele), domain deletion","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus in vivo genetics, multiple orthogonal methods in single rigorous study","pmids":["26255844"],"is_preprint":false},{"year":2006,"finding":"Dia2 forms an SCF E3 ubiquitin ligase complex (SCF(Dia2)) in budding yeast and binds replication origins; deletion of DIA2 causes premature S-phase entry and DNA damage accumulation in S and G2/M phases; these defects require the F-box domain.","method":"Genetic deletion, cell cycle analysis, chromatin immunoprecipitation","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cell-cycle phenotype plus ChIP, single lab","pmids":["16421250"],"is_preprint":false},{"year":2006,"finding":"Dia2 is required for stable passage of replication forks through damaged DNA and natural fragile regions (especially the rDNA replication fork barrier); loss of Dia2 activates the Rad53 checkpoint kinase, elevates DNA repair foci, and increases gross chromosomal rearrangements and extrachromosomal rDNA circles.","method":"Genetic deletion, systematic synthetic lethal screen (~4400 deletion mutants), checkpoint kinase assay, GCR assay","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods across genome-wide genetic screen plus defined molecular phenotypes, replicated","pmids":["16751663"],"is_preprint":false},{"year":2007,"finding":"Yra1 (an mRNA export protein) is a Dia2 interaction partner; Yra1 and Dia2 co-bind replication origins and co-immunoprecipitate with DNA polymerase delta subunit Hys2; Dia2 binding to replication origins is significantly reduced when Yra1 association is compromised, suggesting Yra1 recruits Dia2 to chromatin.","method":"Affinity purification, co-immunoprecipitation, ChIP","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus ChIP with genetic validation, single lab","pmids":["17452447"],"is_preprint":false},{"year":2010,"finding":"Dia2 is itself targeted for ubiquitin-mediated proteolysis; activation of the S-phase checkpoint inhibits Dia2 protein degradation; an N-terminal domain (also required for nuclear localization) is necessary for degradation; Dia2 degradation does not involve an autocatalytic F-box mechanism.","method":"Protein stability assays, checkpoint mutant analysis, domain deletion, NLS addition rescue","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple domain mutants and genetic backgrounds, single lab","pmids":["19858292"],"is_preprint":false},{"year":2012,"finding":"The Hect-domain E3 ubiquitin ligase Tom1 ubiquitinates and degrades Dia2 during G1 and G2/M phases of the cell cycle; Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase (when Dia2 is stabilized); Tom1 recognizes specific positively charged residues in the Dia2 degradation/NLS domain; loss of these residues blocks Tom1-mediated turnover and causes a G1-to-S delay.","method":"In vivo ubiquitination assay, co-immunoprecipitation, cell cycle staging, domain mutagenesis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vivo ubiquitination plus domain mutagenesis plus cell-cycle phenotype, single lab with multiple orthogonal methods","pmids":["22933573"],"is_preprint":false},{"year":2012,"finding":"SCF(Dia2) is required for checkpoint recovery from MMS-induced DNA damage: Dia2 promotes deactivation of the Rad53 checkpoint kinase and contributes to Mrc1 degradation during S-phase checkpoint recovery; checkpoint-defective mrc1 alleles suppress the MMS sensitivity and checkpoint recovery defect of dia2Δ cells.","method":"Genetic suppressor screen, Rad53 kinase assay, protein stability assay, MMS sensitivity assay","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus biochemical kinase readout and degradation assay, single lab","pmids":["23172854"],"is_preprint":false},{"year":2012,"finding":"Dia2 and Tom1 each independently bind the C-terminal region of Cdc6 and control Cdc6 ubiquitination and degradation during G1 phase, acting separately from SCF(Cdc4); loss of Dia2 leads to aberrant Cdc6 and Mcm4 chromatin association in G1.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, chromatin fractionation, genetic deletion","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus chromatin fractionation, single lab","pmids":["23129771"],"is_preprint":false},{"year":2011,"finding":"Dia2 is involved in controlling assembly of the RSC chromatin-remodelling complex; in the absence of Dia2, RSC-mediated transcription regulation is impaired with abnormalities in nucleosome positioning.","method":"Genetic interaction screen, transcription assays, nucleosome positioning assay","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic interactions plus transcription phenotype but limited direct mechanistic biochemistry, single lab","pmids":["21701592"],"is_preprint":false},{"year":2019,"finding":"Human DIAPH2 controls spindle microtubule (MT) dynamics during M-phase in colorectal cancer cells independent of Cdc42 activity; DIAPH2 localizes to spindle MTs in metaphase; full-length DIAPH2 mediates a ~10-fold increase in MT polymerization in vitro independent of its FH2-domain actin-nucleating activity; a region outside the FH2 domain constitutes a second MT-binding site with distinct effects on MT dynamics.","method":"Stable knockdown (shRNA), live-cell imaging, in vitro MT polymerization assay, domain deletion mutant (ΔFH2)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution plus cellular imaging and domain mutant analysis, single lab","pmids":["30926831"],"is_preprint":false},{"year":2019,"finding":"Heterozygous DIAPH2 inactivation (CRISPR/Cas9) in HEK-293T cells shifts cells from a proliferative to a migratory phenotype, consistent with increased metastatic potential.","method":"CRISPR/Cas9 gene editing, proliferation and migration assays","journal":"Carcinogenesis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single heterozygous cell line, phenotypic assay without pathway mechanism","pmids":["30793164"],"is_preprint":false},{"year":2023,"finding":"Mouse Diaph2 is expressed in the actin-rich stereocilia of cochlear outer hair cells; a missense variant in DIAPH2 segregating with X-linked hearing loss causes functional impairment of the protein upon RhoA-dependent activation in vitro.","method":"Immunohistochemistry (mouse cochlea), in vitro RhoA-activation assay, CRISPR/Cas9 knock-out/knock-in mice with ABR measurements","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with in vitro functional assay and RhoA-activation readout, single lab","pmids":["36689403"],"is_preprint":false},{"year":2021,"finding":"Absence of Dia2 in S. cerevisiae prolongs both S- and G2/M-phases, activates the S-phase checkpoint, and causes Ctf4 (a Dia2 substrate) to remain bound to chromatin for an extended period during these phases.","method":"Flow cytometry cell cycle analysis, chromatin fractionation/immunoprecipitation in dia2Δ cells","journal":"Molecules (Basel, Switzerland)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single deletion strain with cell-cycle and chromatin phenotype but limited direct mechanistic follow-up, single lab","pmids":["35011329"],"is_preprint":false}],"current_model":"DIAPH2/Dia2 is a formin-family protein (in metazoans) and F-box subunit of the SCF(Dia2) E3 ubiquitin ligase (in budding yeast) that uses an N-terminal TPR domain to tether itself to the replisome progression complex via Mrc1 and Ctf4, thereby ubiquitylating CMG helicase subunit Mcm7 to drive replication termination and CMG disassembly via Cdc48; it also ubiquitylates Mrc1 to promote checkpoint recovery, degrades Cdc6 in G1, and is itself degraded in a cell-cycle-dependent manner by the Hect E3 ligase Tom1 (stabilized during S phase by checkpoint activation); the human/mammalian ortholog additionally regulates spindle microtubule dynamics through a region outside its FH2 domain and localises to stereocilia in cochlear hair cells where RhoA-dependent activation controls its formin activity."},"narrative":{"mechanistic_narrative":"DIAPH2/Dia2 has two distinct, evidence-supported identities in the available corpus: in budding yeast it functions as the F-box substrate-recognition subunit of an SCF(Dia2) E3 ubiquitin ligase that governs DNA replication, while the human ortholog acts as a cytoskeletal regulator [PMID:16421250, PMID:30926831]. In yeast, Dia2 assembles an SCF complex through its F-box domain and binds replication origins, with its N-terminal TPR domain tethering the ligase to the replisome progression complex via the components Mrc1 and Ctf4 to concentrate it at replication forks [PMID:19913425, PMID:16421250]. So positioned, SCF(Dia2) ubiquitylates the Mcm7 subunit of the CMG helicase at the end of replication to drive replisome disassembly through the Cdc48 segregase, the LRR motif being required for regulation of fork progression [PMID:19910927, PMID:26255844]. Dia2 supports stable fork passage through damaged DNA and fragile regions such as the rDNA replication fork barrier, restrains premature S-phase entry, and promotes checkpoint recovery by aiding Rad53 deactivation and Mrc1 degradation; it additionally degrades Cdc6 in G1 to control origin licensing [PMID:16751663, PMID:23172854, PMID:23129771]. Dia2 is itself cell-cycle-regulated: the Hect E3 ligase Tom1 ubiquitylates and degrades it during G1 and G2/M via positively charged residues in its N-terminal degradation/NLS domain, while S-phase checkpoint activation stabilizes it [PMID:19858292, PMID:22933573]. In a separate functional arena, human DIAPH2 localizes to spindle microtubules and promotes microtubule polymerization through a microtubule-binding region outside its FH2 actin-nucleating domain, and mouse Diaph2 is expressed in cochlear outer hair cell stereocilia where a RhoA-activatable missense variant segregating with X-linked hearing loss impairs its function [PMID:30926831, PMID:36689403].","teleology":[{"year":2006,"claim":"Establishing that Dia2 acts as an SCF E3 ligase subunit at replication origins reframed it from an uncharacterized factor into a cell-cycle regulator of replication and genome stability.","evidence":"Genetic deletion, cell-cycle analysis, and ChIP showing SCF assembly, origin binding, premature S-phase entry, and F-box-dependent damage phenotypes in budding yeast","pmids":["16421250","16751663"],"confidence":"High","gaps":["Direct ubiquitylation substrates not yet identified at this stage","Mechanism linking origin binding to fork stability undefined"]},{"year":2007,"claim":"Identifying Yra1 as a recruiter addressed how Dia2 is delivered to chromatin.","evidence":"Affinity purification, reciprocal Co-IP, and ChIP showing Yra1-dependent origin recruitment of Dia2 and co-binding with Pol-delta subunit Hys2","pmids":["17452447"],"confidence":"Medium","gaps":["Single-lab interaction","Relationship between Yra1- and TPR/replisome-mediated recruitment unresolved"]},{"year":2009,"claim":"Domain mapping resolved how Dia2 is physically coupled to the replisome and which motifs carry out distinct functions, defining the architecture of fork-associated ubiquitylation.","evidence":"Reciprocal Co-IP, two-hybrid, in vitro and in vivo ubiquitination, and domain deletion in two concurrent yeast studies showing TPR-Mrc1/Ctf4 tethering and LRR-dependent fork regulation","pmids":["19913425","19910927"],"confidence":"High","gaps":["The decisive in vivo substrate driving fork regulation not pinned down here","Functional consequence of Mrc1/Ctf4 ubiquitylation ambiguous"]},{"year":2010,"claim":"Showing Dia2 is itself a degradation target under checkpoint control established a feedback layer regulating ligase abundance across the cell cycle.","evidence":"Protein stability assays, checkpoint-mutant analysis, and domain deletion identifying an N-terminal degradation/NLS domain and checkpoint-dependent stabilization","pmids":["19858292"],"confidence":"Medium","gaps":["The responsible E3 ligase not identified at this stage","Single lab"]},{"year":2012,"claim":"Identifying Tom1 as the E3 that degrades Dia2, and Dia2/Tom1 roles in Cdc6 turnover and checkpoint recovery, connected ligase regulation to G1 origin licensing and damage response.","evidence":"In vivo ubiquitination, Co-IP, chromatin fractionation, kinase assays, and genetic suppression across three yeast studies","pmids":["22933573","23129771","23172854"],"confidence":"High","gaps":["Whether Mrc1 degradation in recovery is direct vs indirect not fully resolved","Tom1 substrate-recognition structural basis undefined"]},{"year":2015,"claim":"Reconstituting Mcm7 ubiquitylation defined the central terminal-replication function: SCF(Dia2) triggers CMG helicase disassembly via Cdc48.","evidence":"In vitro and in vivo ubiquitylation, synthetic lethality with cdc48 allele, and TPR-tethering domain analysis showing Mcm7 as the key substrate, while ruling out replisome-specific Mrc1/Ctf4 degradation","pmids":["26255844"],"confidence":"High","gaps":["How Mcm7 is selected at replication termination unresolved","Conservation of this pathway to the metazoan ortholog untested"]},{"year":2019,"claim":"Characterizing human DIAPH2 in mitosis revealed an FH2-independent microtubule-regulating activity, distinct from the yeast ubiquitin-ligase role.","evidence":"shRNA knockdown, live-cell imaging, in vitro MT polymerization assay, and a delta-FH2 mutant in colorectal cancer cells","pmids":["30926831","30793164"],"confidence":"Medium","gaps":["Molecular partners of the non-FH2 MT-binding region unknown","Relationship between MT activity and migratory phenotype not mechanistically linked"]},{"year":2023,"claim":"Demonstrating stereocilia expression and a RhoA-activatable disease variant tied human DIAPH2 to actin-based sensory hair-cell function and X-linked hearing loss.","evidence":"Mouse cochlear immunohistochemistry, in vitro RhoA-activation functional assay, and CRISPR knockout/knockin mice with ABR measurements","pmids":["36689403"],"confidence":"Medium","gaps":["Downstream cytoskeletal effectors in hair cells not identified","Single lab"]},{"year":null,"claim":"Whether the yeast SCF/replication functions and the metazoan cytoskeletal/formin functions reflect divergent activities of one conserved protein or fundamentally distinct proteins remains unresolved in the corpus.","evidence":"No cross-system study reconciling the ubiquitin-ligase and microtubule/actin roles","pmids":[],"confidence":"Low","gaps":["No experiment tests both activities in a single ortholog","Domain conservation between yeast Dia2 and human DIAPH2 not addressed in timeline"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,3]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,4]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[12]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[14]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[3,5]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4,10]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,8]}],"complexes":["SCF(Dia2)","replisome progression complex (associated)"],"partners":["MRC1","CTF4","MCM7","CDC48","TOM1","CDC6","YRA1","RHOA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60879","full_name":"Protein diaphanous homolog 2","aliases":["Diaphanous-related formin-2","DRF2"],"length_aa":1101,"mass_kda":125.6,"function":"Could be involved in oogenesis. Involved in the regulation of endosome dynamics. Implicated in a novel signal transduction pathway, in which isoform 3 and CSK are sequentially activated by RHOD to regulate the motility of early endosomes through interactions with the actin cytoskeleton","subcellular_location":"Cytoplasm, cytosol; Early endosome","url":"https://www.uniprot.org/uniprotkb/O60879/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DIAPH2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ESD","stoichiometry":0.2},{"gene":"PFN1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DIAPH2","total_profiled":1310},"omim":[{"mim_id":"614567","title":"DIAPHANOUS-RELATED FORMIN 3; DIAPH3","url":"https://www.omim.org/entry/614567"},{"mim_id":"605781","title":"RAS HOMOLOG GENE FAMILY, MEMBER D; RHOD","url":"https://www.omim.org/entry/605781"},{"mim_id":"602121","title":"DIAPHANOUS-RELATED FORMIN 1; DIAPH1","url":"https://www.omim.org/entry/602121"},{"mim_id":"311360","title":"PREMATURE OVARIAN FAILURE 1; POF1","url":"https://www.omim.org/entry/311360"},{"mim_id":"300511","title":"PREMATURE OVARIAN FAILURE 2A; POF2A","url":"https://www.omim.org/entry/300511"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Vesicles","reliability":"Supported"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DIAPH2"},"hgnc":{"alias_symbol":["POF","DIA","POF2","DIA2"],"prev_symbol":[]},"alphafold":{"accession":"O60879","domains":[{"cath_id":"1.10.20.40","chopping":"97-149","consensus_level":"medium","plddt":82.0634,"start":97,"end":149},{"cath_id":"1.20.58.2220","chopping":"712-816_914-966","consensus_level":"medium","plddt":90.5141,"start":712,"end":966},{"cath_id":"1.20.5","chopping":"479-529","consensus_level":"medium","plddt":77.5486,"start":479,"end":529}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60879","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60879-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60879-F1-predicted_aligned_error_v6.png","plddt_mean":75.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DIAPH2","jax_strain_url":"https://www.jax.org/strain/search?query=DIAPH2"},"sequence":{"accession":"O60879","fasta_url":"https://rest.uniprot.org/uniprotkb/O60879.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60879/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60879"}},"corpus_meta":[{"pmid":"19913425","id":"PMC_19913425","title":"The 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/26367735","citation_count":2,"is_preprint":false},{"pmid":"35011329","id":"PMC_35011329","title":"The Effect of Dia2 Protein Deficiency on the Cell Cycle, Cell Size, and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae.","date":"2021","source":"Molecules (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/35011329","citation_count":1,"is_preprint":false},{"pmid":"38844723","id":"PMC_38844723","title":"DIAPH2 gene polymorphisms and laryngeal cancer risk in men.","date":"2024","source":"Journal of cancer research and clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38844723","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.31.646288","title":"<i>In vivo</i>  activation of the  <i>dia</i>  BGC allows consolidation of the biosynthetic pathways of diaporthin, dichlorodiaporthin, diaporthinic acid, and diaporthinol","date":"2025-04-01","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.31.646288","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14402,"output_tokens":3609,"usd":0.04867,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11301,"output_tokens":3269,"usd":0.069115,"stage2_stop_reason":"end_turn"},"total_usd":0.117785,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"The TPR domain at the amino terminus of yeast Dia2 tethers SCF(Dia2) to the replisome progression complex (RPC) by interacting with the RPC components Mrc1 and Ctf4, increasing the local concentration of the E3 ligase at DNA replication forks.\",\n      \"method\": \"Co-immunoprecipitation, two-hybrid interaction, domain deletion analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with domain mapping, replicated independently in two concurrent papers (PMID 19913425 and PMID 19910927)\",\n      \"pmids\": [\"19913425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SCF(Dia2) ubiquitinates replisome components Mrc1 and Ctf4 both in vivo and in vitro, and the leucine-rich repeat (LRR) motif of Dia2 is required for regulation of replisome progression while the TPR motif mediates interaction with replisome components and Dia2 stability.\",\n      \"method\": \"In vitro ubiquitination assay, in vivo ubiquitination, domain deletion analysis, yeast two-hybrid\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination assay plus domain mutagenesis, replicated in companion paper\",\n      \"pmids\": [\"19910927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Dia2 localizes to replication forks and regulates replication fork progression under hydroxyurea treatment, as shown by ChIP-on-chip analysis.\",\n      \"method\": \"ChIP-on-chip\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide ChIP with functional context, single lab\",\n      \"pmids\": [\"19910927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SCF(Dia2) drives ubiquitylation of the Mcm7 subunit of the CMG helicase at the end of replication, and tethering via the TPR domain to the replisome progression complex increases the efficiency of this ubiquitylation both in vitro and in vivo, promoting CMG disassembly through a pathway requiring the Cdc48 segregase. SCF(Dia2) does NOT mediate replisome-specific degradation of Mrc1 and Ctf4.\",\n      \"method\": \"In vitro ubiquitylation assay, in vivo ubiquitylation, genetic epistasis (synthetic lethality with cdc48 allele), domain deletion\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus in vivo genetics, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"26255844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Dia2 forms an SCF E3 ubiquitin ligase complex (SCF(Dia2)) in budding yeast and binds replication origins; deletion of DIA2 causes premature S-phase entry and DNA damage accumulation in S and G2/M phases; these defects require the F-box domain.\",\n      \"method\": \"Genetic deletion, cell cycle analysis, chromatin immunoprecipitation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cell-cycle phenotype plus ChIP, single lab\",\n      \"pmids\": [\"16421250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Dia2 is required for stable passage of replication forks through damaged DNA and natural fragile regions (especially the rDNA replication fork barrier); loss of Dia2 activates the Rad53 checkpoint kinase, elevates DNA repair foci, and increases gross chromosomal rearrangements and extrachromosomal rDNA circles.\",\n      \"method\": \"Genetic deletion, systematic synthetic lethal screen (~4400 deletion mutants), checkpoint kinase assay, GCR assay\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods across genome-wide genetic screen plus defined molecular phenotypes, replicated\",\n      \"pmids\": [\"16751663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Yra1 (an mRNA export protein) is a Dia2 interaction partner; Yra1 and Dia2 co-bind replication origins and co-immunoprecipitate with DNA polymerase delta subunit Hys2; Dia2 binding to replication origins is significantly reduced when Yra1 association is compromised, suggesting Yra1 recruits Dia2 to chromatin.\",\n      \"method\": \"Affinity purification, co-immunoprecipitation, ChIP\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus ChIP with genetic validation, single lab\",\n      \"pmids\": [\"17452447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Dia2 is itself targeted for ubiquitin-mediated proteolysis; activation of the S-phase checkpoint inhibits Dia2 protein degradation; an N-terminal domain (also required for nuclear localization) is necessary for degradation; Dia2 degradation does not involve an autocatalytic F-box mechanism.\",\n      \"method\": \"Protein stability assays, checkpoint mutant analysis, domain deletion, NLS addition rescue\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple domain mutants and genetic backgrounds, single lab\",\n      \"pmids\": [\"19858292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The Hect-domain E3 ubiquitin ligase Tom1 ubiquitinates and degrades Dia2 during G1 and G2/M phases of the cell cycle; Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase (when Dia2 is stabilized); Tom1 recognizes specific positively charged residues in the Dia2 degradation/NLS domain; loss of these residues blocks Tom1-mediated turnover and causes a G1-to-S delay.\",\n      \"method\": \"In vivo ubiquitination assay, co-immunoprecipitation, cell cycle staging, domain mutagenesis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vivo ubiquitination plus domain mutagenesis plus cell-cycle phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22933573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SCF(Dia2) is required for checkpoint recovery from MMS-induced DNA damage: Dia2 promotes deactivation of the Rad53 checkpoint kinase and contributes to Mrc1 degradation during S-phase checkpoint recovery; checkpoint-defective mrc1 alleles suppress the MMS sensitivity and checkpoint recovery defect of dia2Δ cells.\",\n      \"method\": \"Genetic suppressor screen, Rad53 kinase assay, protein stability assay, MMS sensitivity assay\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus biochemical kinase readout and degradation assay, single lab\",\n      \"pmids\": [\"23172854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Dia2 and Tom1 each independently bind the C-terminal region of Cdc6 and control Cdc6 ubiquitination and degradation during G1 phase, acting separately from SCF(Cdc4); loss of Dia2 leads to aberrant Cdc6 and Mcm4 chromatin association in G1.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, chromatin fractionation, genetic deletion\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus chromatin fractionation, single lab\",\n      \"pmids\": [\"23129771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Dia2 is involved in controlling assembly of the RSC chromatin-remodelling complex; in the absence of Dia2, RSC-mediated transcription regulation is impaired with abnormalities in nucleosome positioning.\",\n      \"method\": \"Genetic interaction screen, transcription assays, nucleosome positioning assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic interactions plus transcription phenotype but limited direct mechanistic biochemistry, single lab\",\n      \"pmids\": [\"21701592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human DIAPH2 controls spindle microtubule (MT) dynamics during M-phase in colorectal cancer cells independent of Cdc42 activity; DIAPH2 localizes to spindle MTs in metaphase; full-length DIAPH2 mediates a ~10-fold increase in MT polymerization in vitro independent of its FH2-domain actin-nucleating activity; a region outside the FH2 domain constitutes a second MT-binding site with distinct effects on MT dynamics.\",\n      \"method\": \"Stable knockdown (shRNA), live-cell imaging, in vitro MT polymerization assay, domain deletion mutant (ΔFH2)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution plus cellular imaging and domain mutant analysis, single lab\",\n      \"pmids\": [\"30926831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Heterozygous DIAPH2 inactivation (CRISPR/Cas9) in HEK-293T cells shifts cells from a proliferative to a migratory phenotype, consistent with increased metastatic potential.\",\n      \"method\": \"CRISPR/Cas9 gene editing, proliferation and migration assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single heterozygous cell line, phenotypic assay without pathway mechanism\",\n      \"pmids\": [\"30793164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Mouse Diaph2 is expressed in the actin-rich stereocilia of cochlear outer hair cells; a missense variant in DIAPH2 segregating with X-linked hearing loss causes functional impairment of the protein upon RhoA-dependent activation in vitro.\",\n      \"method\": \"Immunohistochemistry (mouse cochlea), in vitro RhoA-activation assay, CRISPR/Cas9 knock-out/knock-in mice with ABR measurements\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with in vitro functional assay and RhoA-activation readout, single lab\",\n      \"pmids\": [\"36689403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Absence of Dia2 in S. cerevisiae prolongs both S- and G2/M-phases, activates the S-phase checkpoint, and causes Ctf4 (a Dia2 substrate) to remain bound to chromatin for an extended period during these phases.\",\n      \"method\": \"Flow cytometry cell cycle analysis, chromatin fractionation/immunoprecipitation in dia2Δ cells\",\n      \"journal\": \"Molecules (Basel, Switzerland)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single deletion strain with cell-cycle and chromatin phenotype but limited direct mechanistic follow-up, single lab\",\n      \"pmids\": [\"35011329\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DIAPH2/Dia2 is a formin-family protein (in metazoans) and F-box subunit of the SCF(Dia2) E3 ubiquitin ligase (in budding yeast) that uses an N-terminal TPR domain to tether itself to the replisome progression complex via Mrc1 and Ctf4, thereby ubiquitylating CMG helicase subunit Mcm7 to drive replication termination and CMG disassembly via Cdc48; it also ubiquitylates Mrc1 to promote checkpoint recovery, degrades Cdc6 in G1, and is itself degraded in a cell-cycle-dependent manner by the Hect E3 ligase Tom1 (stabilized during S phase by checkpoint activation); the human/mammalian ortholog additionally regulates spindle microtubule dynamics through a region outside its FH2 domain and localises to stereocilia in cochlear hair cells where RhoA-dependent activation controls its formin activity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DIAPH2/Dia2 has two distinct, evidence-supported identities in the available corpus: in budding yeast it functions as the F-box substrate-recognition subunit of an SCF(Dia2) E3 ubiquitin ligase that governs DNA replication, while the human ortholog acts as a cytoskeletal regulator [#4, #12]. In yeast, Dia2 assembles an SCF complex through its F-box domain and binds replication origins, with its N-terminal TPR domain tethering the ligase to the replisome progression complex via the components Mrc1 and Ctf4 to concentrate it at replication forks [#0, #4]. So positioned, SCF(Dia2) ubiquitylates the Mcm7 subunit of the CMG helicase at the end of replication to drive replisome disassembly through the Cdc48 segregase, the LRR motif being required for regulation of fork progression [#1, #3]. Dia2 supports stable fork passage through damaged DNA and fragile regions such as the rDNA replication fork barrier, restrains premature S-phase entry, and promotes checkpoint recovery by aiding Rad53 deactivation and Mrc1 degradation; it additionally degrades Cdc6 in G1 to control origin licensing [#5, #9, #10]. Dia2 is itself cell-cycle-regulated: the Hect E3 ligase Tom1 ubiquitylates and degrades it during G1 and G2/M via positively charged residues in its N-terminal degradation/NLS domain, while S-phase checkpoint activation stabilizes it [#7, #8]. In a separate functional arena, human DIAPH2 localizes to spindle microtubules and promotes microtubule polymerization through a microtubule-binding region outside its FH2 actin-nucleating domain, and mouse Diaph2 is expressed in cochlear outer hair cell stereocilia where a RhoA-activatable missense variant segregating with X-linked hearing loss impairs its function [#12, #14].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that Dia2 acts as an SCF E3 ligase subunit at replication origins reframed it from an uncharacterized factor into a cell-cycle regulator of replication and genome stability.\",\n      \"evidence\": \"Genetic deletion, cell-cycle analysis, and ChIP showing SCF assembly, origin binding, premature S-phase entry, and F-box-dependent damage phenotypes in budding yeast\",\n      \"pmids\": [\"16421250\", \"16751663\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitylation substrates not yet identified at this stage\", \"Mechanism linking origin binding to fork stability undefined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identifying Yra1 as a recruiter addressed how Dia2 is delivered to chromatin.\",\n      \"evidence\": \"Affinity purification, reciprocal Co-IP, and ChIP showing Yra1-dependent origin recruitment of Dia2 and co-binding with Pol-delta subunit Hys2\",\n      \"pmids\": [\"17452447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab interaction\", \"Relationship between Yra1- and TPR/replisome-mediated recruitment unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Domain mapping resolved how Dia2 is physically coupled to the replisome and which motifs carry out distinct functions, defining the architecture of fork-associated ubiquitylation.\",\n      \"evidence\": \"Reciprocal Co-IP, two-hybrid, in vitro and in vivo ubiquitination, and domain deletion in two concurrent yeast studies showing TPR-Mrc1/Ctf4 tethering and LRR-dependent fork regulation\",\n      \"pmids\": [\"19913425\", \"19910927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The decisive in vivo substrate driving fork regulation not pinned down here\", \"Functional consequence of Mrc1/Ctf4 ubiquitylation ambiguous\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showing Dia2 is itself a degradation target under checkpoint control established a feedback layer regulating ligase abundance across the cell cycle.\",\n      \"evidence\": \"Protein stability assays, checkpoint-mutant analysis, and domain deletion identifying an N-terminal degradation/NLS domain and checkpoint-dependent stabilization\",\n      \"pmids\": [\"19858292\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The responsible E3 ligase not identified at this stage\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying Tom1 as the E3 that degrades Dia2, and Dia2/Tom1 roles in Cdc6 turnover and checkpoint recovery, connected ligase regulation to G1 origin licensing and damage response.\",\n      \"evidence\": \"In vivo ubiquitination, Co-IP, chromatin fractionation, kinase assays, and genetic suppression across three yeast studies\",\n      \"pmids\": [\"22933573\", \"23129771\", \"23172854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Mrc1 degradation in recovery is direct vs indirect not fully resolved\", \"Tom1 substrate-recognition structural basis undefined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Reconstituting Mcm7 ubiquitylation defined the central terminal-replication function: SCF(Dia2) triggers CMG helicase disassembly via Cdc48.\",\n      \"evidence\": \"In vitro and in vivo ubiquitylation, synthetic lethality with cdc48 allele, and TPR-tethering domain analysis showing Mcm7 as the key substrate, while ruling out replisome-specific Mrc1/Ctf4 degradation\",\n      \"pmids\": [\"26255844\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Mcm7 is selected at replication termination unresolved\", \"Conservation of this pathway to the metazoan ortholog untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Characterizing human DIAPH2 in mitosis revealed an FH2-independent microtubule-regulating activity, distinct from the yeast ubiquitin-ligase role.\",\n      \"evidence\": \"shRNA knockdown, live-cell imaging, in vitro MT polymerization assay, and a delta-FH2 mutant in colorectal cancer cells\",\n      \"pmids\": [\"30926831\", \"30793164\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular partners of the non-FH2 MT-binding region unknown\", \"Relationship between MT activity and migratory phenotype not mechanistically linked\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrating stereocilia expression and a RhoA-activatable disease variant tied human DIAPH2 to actin-based sensory hair-cell function and X-linked hearing loss.\",\n      \"evidence\": \"Mouse cochlear immunohistochemistry, in vitro RhoA-activation functional assay, and CRISPR knockout/knockin mice with ABR measurements\",\n      \"pmids\": [\"36689403\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream cytoskeletal effectors in hair cells not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether the yeast SCF/replication functions and the metazoan cytoskeletal/formin functions reflect divergent activities of one conserved protein or fundamentally distinct proteins remains unresolved in the corpus.\",\n      \"evidence\": \"No cross-system study reconciling the ubiquitin-ligase and microtubule/actin roles\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experiment tests both activities in a single ortholog\", \"Domain conservation between yeast Dia2 and human DIAPH2 not addressed in timeline\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 10]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"complexes\": [\"SCF(Dia2)\", \"replisome progression complex (associated)\"],\n    \"partners\": [\"Mrc1\", \"Ctf4\", \"Mcm7\", \"Cdc48\", \"Tom1\", \"Cdc6\", \"Yra1\", \"RhoA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}