{"gene":"RFC4","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1994,"finding":"RFC4 (yeast Rfc4p) encodes the 37-kDa subunit of replication factor C and is essential for yeast viability. Purified Rfc4p formed a tight complex with the Rfc3p subunit (the ATPase of RFC), establishing a direct physical interaction between these two small RFC subunits.","method":"Cloning, overexpression in E. coli, purification, biochemical characterization, protein-protein interaction assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical demonstration of Rfc4p-Rfc3p complex formation in single study; essential gene established by genetics","pmids":["8063832"],"is_preprint":false},{"year":2001,"finding":"The ATP-binding domain of Rfc4 (Walker A motif, K55) is essential for DNA recognition and clamp loading by RFC. The rfc4-K55E mutant complex retained PCNA interaction and clamp loading activity but only at very high ATP concentrations, indicating Rfc4's ATPase domain contributes to ATP-dependent clamp loading.","method":"Site-directed mutagenesis of Walker A motif, overproduction of mutant RFC complexes in E. coli, in vitro ATPase assay, clamp loading assay, DNA binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple mutant complexes tested with orthogonal biochemical assays in a single rigorous study","pmids":["11432854"],"is_preprint":false},{"year":2001,"finding":"Yeast Rfc4 physically interacts with the N-terminal domain of Rpa1 (Rpa1N), and this interaction is required for both DNA replication and DNA damage checkpoint function. rfc4-2 is synthetically lethal with rfa1-t11 (an Rpa1N mutation). Rfc4 functions as a sensor in the G1/S DNA damage checkpoint, intra-S checkpoint (replication block), and G2/M DNA damage checkpoint, and is epistatic with RAD24 for DNA damage sensitivity.","method":"Yeast two-hybrid/allele-specific genetic interaction (synthetic lethality screen), hydroxyurea sensitivity, DNA damage checkpoint assays (G1/S, intra-S, G2/M), epistasis analysis with rad24","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal allele-specific genetic interactions, multiple checkpoint pathway epistasis tests, and functional checkpoint readouts across multiple assays in single study","pmids":["11340166"],"is_preprint":false},{"year":2001,"finding":"Drosophila Rfc4 (DmRfc4) protein localizes to all replicating nuclei but is dispersed from chromatin during mitosis. Loss-of-function mutations in DmRfc4 cause defects in the DNA replication block and DNA damage checkpoints (not the kinetochore attachment checkpoint), leading to aberrant mitotic chromosome condensation and premature sister chromatid separation.","method":"Immunofluorescence localization in larval tissue, genetic loss-of-function analysis of two alleles, checkpoint assays with DNA replication inhibitors and DNA-damaging agents","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct protein localization by immunofluorescence tied to functional consequence, two independent alleles, multiple checkpoint assays; consistent with yeast findings","pmids":["11438670"],"is_preprint":false},{"year":1995,"finding":"The human RFC4 gene (encoding the p37 subunit of replication factor C) was mapped to chromosome band 3q27 by PCR from somatic hybrid DNA panels and fluorescence in situ hybridization.","method":"PCR amplification from somatic cell hybrid DNA panel, fluorescence in situ hybridization (FISH)","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct chromosomal mapping by two orthogonal methods, single study","pmids":["7774928"],"is_preprint":false},{"year":2019,"finding":"RFC4 promotes nonhomologous end joining (NHEJ)-mediated DNA repair in colorectal cancer cells by physically interacting with Ku70/Ku80. RFC4 knockdown increased X-ray-induced DNA damage and apoptosis, while RFC4 did not affect homologous recombination-mediated repair.","method":"Genome-wide RNAi screen, RFC4 knockdown/overexpression, Co-immunoprecipitation with Ku70/Ku80, DNA damage assays, apoptosis assay, NHEJ and HR repair assays in vitro and xenograft mouse model","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing Ku70/Ku80 interaction, functional KD/OE with defined repair pathway readout, single lab","pmids":["30979744"],"is_preprint":false},{"year":2021,"finding":"RFC4 directly binds to the Notch1 intracellular domain (NICD1) to competitively block CDK8/FBXW7-mediated ubiquitin-proteasomal degradation of NICD1, thereby stabilizing NICD1. RFC4 is itself a transcriptional target of Notch1 signaling, forming a positive feedback loop. This RFC4–NICD1 interaction promotes NSCLC metastasis and cancer stem cell properties.","method":"Co-immunoprecipitation, competitive binding assays, transcriptional reporter assays, RFC4 overexpression/knockdown, in vitro and in vivo tumor assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing direct RFC4-NICD1 interaction and functional consequences, single lab, multiple orthogonal methods","pmids":["33976158"],"is_preprint":false},{"year":2024,"finding":"Bi-allelic loss-of-function variants in RFC4 that disrupt the C-terminal domain cause destabilization and reduced expression of RFC4 protein, compromised stability of other RFC complex subunits (RFC1–5), and perturbed RFC complex formation. Cell cycle studies using RFC4-deficient HeLa cells and primary fibroblasts showed perturbation of DNA replication and cell cycle progression. Structural analysis of the cryo-EM RFC-PCNA complex suggested the variants disrupt interactions within RFC4 and/or destabilize the RFC complex.","method":"Patient-derived fibroblasts and RFC4-deficient HeLa cells, Western blot for RFC complex subunit stability, RFC complex formation assay, cryo-EM structural analysis (previously determined structure), cell cycle analysis","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cellular functional studies in patient-derived cells with multiple orthogonal methods (protein stability, complex formation, cell cycle), structural analysis; single lab","pmids":["39106866"],"is_preprint":false},{"year":2026,"finding":"TMZ-induced chromatin accessibility allows transcription factor YY1 to bind the RFC4 promoter and upregulate RFC4 expression in GBM. RFC4 then stabilizes the kinase STK38, and the RFC4-STK38 interaction facilitates BECN1 recruitment, activating autophagy and conferring temozolomide resistance. Phosphorylation of STK38 at T444 stabilizes the RFC4-STK38-BECN1 complex; a T444 phospho-deficient mutant impairs autophagy.","method":"Chromatin accessibility assay, ChIP/reporter assay for YY1 binding, Co-immunoprecipitation of RFC4-STK38-BECN1, phospho-deficient mutagenesis, autophagy assays, in vivo xenograft with RFC4 overexpression and autophagy inhibition","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing RFC4-STK38-BECN1 complex, phospho-deficient mutagenesis, multiple functional assays, single lab","pmids":["41872171"],"is_preprint":false},{"year":2026,"finding":"RFC4 silencing in colorectal cancer cells suppresses proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and Wnt/β-catenin signaling pathway activity. The Wnt/β-catenin pathway activator BML-284 partially reversed the inhibitory effects of RFC4 silencing on invasion and EMT, positioning RFC4 upstream of Wnt/β-catenin activation.","method":"RFC4 siRNA knockdown, proliferation/migration/invasion assays, EMT marker Western blot, Wnt/β-catenin pathway reporter/western blot, BML-284 rescue experiment, in vivo liver metastasis mouse model","journal":"Cell biology international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — epistasis established by pharmacological rescue, single lab, no direct binding between RFC4 and Wnt pathway components demonstrated","pmids":["41574400"],"is_preprint":false},{"year":2026,"finding":"The small molecule Platycodin D (PD) binds to RFC4 (identified by thermal proteome profiling, CETSA, and PELSA), and the PD-RFC4 complex reduces nuclear entry of Notch1 and Notch3 intracellular domains, promoting their degradation via ubiquitination and downregulating Notch signaling.","method":"Thermal proteome profiling (TPP), molecular docking, CETSA, PELSA, Western blot, immunoprecipitation-Western blot (IP-WB), proteomic and ubiquitinomic profiling","journal":"Molecular & cellular proteomics","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal biophysical binding assays (TPP, CETSA, PELSA) establishing direct RFC4-PD interaction, downstream signaling validated by IP-WB and proteomics; single lab","pmids":["42142580"],"is_preprint":false}],"current_model":"RFC4 is a conserved subunit of the replication factor C (RFC) clamp loader complex whose ATP-binding domain (Walker A motif K55) is required for DNA-dependent ATPase activity and PCNA clamp loading onto DNA; it directly interacts with Rpa1N to serve as a sensor in multiple DNA damage and replication checkpoints (G1/S, intra-S, G2/M); in cancer contexts, RFC4 stabilizes NICD1 by blocking CDK8/FBXW7-mediated degradation to activate Notch signaling, promotes NHEJ-mediated DNA repair through Ku70/Ku80 interaction, and drives autophagy-based chemoresistance by stabilizing the STK38-BECN1 complex."},"narrative":{"mechanistic_narrative":"RFC4 is a conserved subunit of the replication factor C (RFC) clamp-loader complex that couples ATP-dependent recognition of primer-template DNA to loading of the PCNA sliding clamp during DNA replication [PMID:8063832, PMID:11432854]. Its ATPase domain is functionally essential: the Walker A K55 residue is required for efficient DNA-dependent clamp loading, as a K55E mutant complex loads PCNA only at very high ATP concentrations [PMID:11432854]. Beyond catalysis, RFC4 physically interacts with the N-terminal domain of Rpa1 (Rpa1N), and this contact is required for both replication and the G1/S, intra-S, and G2/M DNA damage checkpoints, placing RFC4 as a sensor that is epistatic with RAD24 [PMID:11340166]; consistent with this, RFC4 localizes to replicating nuclei and its loss causes checkpoint failure with aberrant chromosome condensation and premature sister chromatid separation [PMID:11438670]. RFC4 is required to maintain the integrity and expression of the assembled RFC complex, and bi-allelic loss-of-function variants disrupting its C-terminal domain destabilize RFC4 and the other RFC subunits and perturb DNA replication and cell-cycle progression, defining RFC4 as the basis of a human Mendelian disorder [PMID:39106866]. In cancer settings RFC4 acts in additional protein-stabilizing roles: it promotes NHEJ-mediated repair through interaction with Ku70/Ku80 [PMID:30979744], directly binds NICD1 to block CDK8/FBXW7-mediated degradation and sustain Notch signaling in a positive feedback loop [PMID:33976158], and stabilizes an STK38-BECN1 complex to drive autophagy-based temozolomide resistance [PMID:41872171]. The small molecule Platycodin D binds RFC4 and reduces nuclear Notch intracellular domain accumulation, downregulating Notch signaling [PMID:42142580].","teleology":[{"year":1994,"claim":"Established RFC4 as an essential structural subunit of the RFC clamp-loader and defined its first direct intra-complex contact, anchoring all later mechanism in a defined protein assembly.","evidence":"Cloning, E. coli expression, purification and interaction assay of yeast Rfc4p with Rfc3p","pmids":["8063832"],"confidence":"Medium","gaps":["Does not resolve stoichiometry or architecture of the full RFC pentamer","No demonstration of clamp-loading activity for Rfc4 itself"]},{"year":2001,"claim":"Showed that RFC4's ATPase domain is mechanistically required for ATP-dependent clamp loading, not merely structural.","evidence":"Walker A K55E site-directed mutagenesis with in vitro ATPase, DNA binding and PCNA loading assays on reconstituted RFC complexes","pmids":["11432854"],"confidence":"High","gaps":["Does not define order of ATP hydrolysis among RFC subunits","No structural model of the K55 mutant in the loading reaction"]},{"year":2001,"claim":"Linked RFC4 to checkpoint signaling by identifying a required Rpa1N interaction and placing RFC4 in the G1/S, intra-S, and G2/M checkpoints epistatic with RAD24.","evidence":"Allele-specific synthetic lethality (rfc4-2 / rfa1-t11), checkpoint and HU-sensitivity assays, RAD24 epistasis in yeast","pmids":["11340166"],"confidence":"High","gaps":["Does not define the molecular signal RFC4 transmits to downstream checkpoint kinases","Interaction mapped genetically/two-hybrid, not structurally"]},{"year":2001,"claim":"Tied RFC4 protein localization to checkpoint function in a metazoan, showing chromatin association during replication and checkpoint-specific (not kinetochore) failures upon loss.","evidence":"Immunofluorescence and loss-of-function alleles in Drosophila with replication-block and DNA-damage checkpoint assays","pmids":["11438670"],"confidence":"High","gaps":["Does not address mammalian-specific checkpoint wiring","Mechanism of chromatin dispersal at mitosis unexplained"]},{"year":1995,"claim":"Mapped the human RFC4 gene to 3q27, enabling later human genetic and disease studies.","evidence":"PCR from somatic hybrid panels and FISH","pmids":["7774928"],"confidence":"Medium","gaps":["No functional information","No disease association at the time"]},{"year":2019,"claim":"Extended RFC4 function beyond replication to DNA double-strand break repair, implicating it in NHEJ through Ku70/Ku80.","evidence":"Genome-wide RNAi screen, Co-IP with Ku70/Ku80, NHEJ vs HR repair and apoptosis assays in colorectal cancer cells and xenografts","pmids":["30979744"],"confidence":"Medium","gaps":["Single lab Co-IP without reciprocal/structural validation","Whether NHEJ role depends on intact RFC complex unclear"]},{"year":2021,"claim":"Revealed a non-replicative protein-stabilization role: RFC4 directly binds NICD1 to block its degradation, sustaining Notch signaling in a feedback loop driving metastasis.","evidence":"Co-IP, competitive binding and reporter assays, knockdown/overexpression, in vitro and in vivo NSCLC tumor assays","pmids":["33976158"],"confidence":"Medium","gaps":["Binding interface on RFC4 not mapped","Whether this requires monomeric RFC4 or the RFC complex is unresolved"]},{"year":2024,"claim":"Established RFC4 as the cause of a human Mendelian disorder by showing C-terminal loss-of-function variants destabilize RFC4 and the whole RFC complex with replication/cell-cycle defects.","evidence":"Patient fibroblasts and RFC4-deficient HeLa cells, Western blot of RFC subunit stability, complex-formation and cell-cycle assays, cryo-EM structural interpretation","pmids":["39106866"],"confidence":"Medium","gaps":["Genotype-phenotype spectrum not fully defined","Direct structural data on patient variants not generated"]},{"year":2026,"claim":"Showed RFC4 is transcriptionally inducible (via YY1) and stabilizes an STK38-BECN1 complex to activate autophagy and confer chemoresistance.","evidence":"Chromatin accessibility/ChIP-reporter for YY1, Co-IP of RFC4-STK38-BECN1, T444 phospho-deficient mutagenesis, autophagy assays and xenografts in GBM","pmids":["41872171"],"confidence":"Medium","gaps":["Direct RFC4-STK38 binding interface unmapped","Single lab; relationship to RFC4's clamp-loader role unaddressed"]},{"year":2026,"claim":"Identified RFC4 as a druggable target whose engagement by Platycodin D suppresses nuclear Notch intracellular domain accumulation and Notch signaling.","evidence":"Thermal proteome profiling, CETSA, PELSA binding assays, IP-WB, proteomic and ubiquitinomic profiling","pmids":["42142580"],"confidence":"Medium","gaps":["Binding site of Platycodin D on RFC4 not defined","Mechanistic link between PD binding and Notch nuclear exclusion not fully resolved"]},{"year":2026,"claim":"Positioned RFC4 upstream of Wnt/beta-catenin-driven invasion and EMT in colorectal cancer.","evidence":"siRNA knockdown with proliferation/invasion/EMT assays and pharmacological BML-284 rescue, liver metastasis model","pmids":["41574400"],"confidence":"Low","gaps":["No direct binding between RFC4 and Wnt pathway components demonstrated","Epistasis rests on pharmacological rescue alone","Single lab, not independently confirmed"]},{"year":null,"claim":"How RFC4's canonical clamp-loader/checkpoint functions mechanistically relate to its many cancer-associated protein-stabilization roles (NICD1, Ku70/80, STK38-BECN1, Wnt) remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Unclear whether non-replicative roles require the intact RFC complex or free RFC4","No shared structural binding interface identified across partners"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[1]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,7]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[6,8]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[1,7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,3,7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,9]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[8]}],"complexes":["Replication factor C (RFC) clamp-loader complex"],"partners":["RFC3","RPA1","KU70","KU80","NICD1","STK38","BECN1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P35249","full_name":"Replication factor C subunit 4","aliases":["Activator 1 37 kDa subunit","A1 37 kDa subunit","Activator 1 subunit 4","Replication factor C 37 kDa subunit","RF-C 37 kDa subunit","RFC37"],"length_aa":363,"mass_kda":39.7,"function":"Subunit of the replication factor C (RFC) complex which acts during elongation of primed DNA templates by DNA polymerases delta and epsilon, and is necessary for ATP-dependent loading of proliferating cell nuclear antigen (PCNA) onto primed DNA. The RFC4 subunit probably functions as a scaffold on which the other complex components can assemble","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P35249/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RFC4","classification":"Common Essential","n_dependent_lines":1150,"n_total_lines":1208,"dependency_fraction":0.9519867549668874},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SSRP1","stoichiometry":4.0},{"gene":"CAPRIN1","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"G3BP2","stoichiometry":0.2},{"gene":"H2AFZ","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"HUS1","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2},{"gene":"PARP1","stoichiometry":0.2},{"gene":"PCNA","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RFC4","total_profiled":1310},"omim":[{"mim_id":"621010","title":"MORIMOTO-RYU-MALICDAN NEUROMUSCULAR SYNDROME; MRMNS","url":"https://www.omim.org/entry/621010"},{"mim_id":"613203","title":"DNA REPLICATION AND SISTER CHROMATID COHESION 1; DSCC1","url":"https://www.omim.org/entry/613203"},{"mim_id":"613202","title":"CHROMOSOME TRANSMISSION FIDELITY FACTOR 8; CHTF8","url":"https://www.omim.org/entry/613202"},{"mim_id":"613201","title":"CHROMOSOME TRANSMISSION FIDELITY FACTOR 18; CHTF18","url":"https://www.omim.org/entry/613201"},{"mim_id":"609534","title":"ATPase FAMILY, AAA DOMAIN-CONTAINING, MEMBER 5; ATAD5","url":"https://www.omim.org/entry/609534"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RFC4"},"hgnc":{"alias_symbol":["A1","RFC37"],"prev_symbol":[]},"alphafold":{"accession":"P35249","domains":[{"cath_id":"3.40.50.300","chopping":"41-199","consensus_level":"high","plddt":84.5986,"start":41,"end":199},{"cath_id":"1.10.8.60","chopping":"205-267","consensus_level":"high","plddt":89.5889,"start":205,"end":267},{"cath_id":"1.20.272.10","chopping":"272-362","consensus_level":"high","plddt":90.3377,"start":272,"end":362}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P35249","model_url":"https://alphafold.ebi.ac.uk/files/AF-P35249-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P35249-F1-predicted_aligned_error_v6.png","plddt_mean":82.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RFC4","jax_strain_url":"https://www.jax.org/strain/search?query=RFC4"},"sequence":{"accession":"P35249","fasta_url":"https://rest.uniprot.org/uniprotkb/P35249.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P35249/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P35249"}},"corpus_meta":[{"pmid":"11340166","id":"PMC_11340166","title":"Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae.","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11340166","citation_count":95,"is_preprint":false},{"pmid":"33976158","id":"PMC_33976158","title":"An RFC4/Notch1 signaling feedback loop promotes NSCLC metastasis and stemness.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33976158","citation_count":92,"is_preprint":false},{"pmid":"30979744","id":"PMC_30979744","title":"Genome-wide RNAi Screening Identifies RFC4 as a Factor That Mediates Radioresistance in Colorectal Cancer by Facilitating Nonhomologous End Joining Repair.","date":"2019","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/30979744","citation_count":62,"is_preprint":false},{"pmid":"11432854","id":"PMC_11432854","title":"ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11432854","citation_count":58,"is_preprint":false},{"pmid":"11438670","id":"PMC_11438670","title":"Loss of cell cycle checkpoint control in Drosophila Rfc4 mutants.","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11438670","citation_count":43,"is_preprint":false},{"pmid":"7774928","id":"PMC_7774928","title":"Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23.","date":"1995","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/7774928","citation_count":40,"is_preprint":false},{"pmid":"8063832","id":"PMC_8063832","title":"Cloning and characterization of the essential Saccharomyces cerevisiae RFC4 gene encoding the 37-kDa subunit of replication factor C.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8063832","citation_count":35,"is_preprint":false},{"pmid":"36562948","id":"PMC_36562948","title":"Knockdown of RFC4 inhibits the cell proliferation of nasopharyngeal carcinoma in vitro and in vivo.","date":"2022","source":"Frontiers of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36562948","citation_count":24,"is_preprint":false},{"pmid":"33783864","id":"PMC_33783864","title":"RFC4 promotes the progression and growth of Oral Tongue squamous cell carcinoma in vivo and vitro.","date":"2021","source":"Journal of clinical laboratory analysis","url":"https://pubmed.ncbi.nlm.nih.gov/33783864","citation_count":18,"is_preprint":false},{"pmid":"39673181","id":"PMC_39673181","title":"Knockdown of RFC4 inhibits cell proliferation of oral squamous cell carcinoma in vitro and in vivo.","date":"2024","source":"FEBS open bio","url":"https://pubmed.ncbi.nlm.nih.gov/39673181","citation_count":6,"is_preprint":false},{"pmid":"39106866","id":"PMC_39106866","title":"Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.","date":"2024","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39106866","citation_count":3,"is_preprint":false},{"pmid":"39672175","id":"PMC_39672175","title":"RFC4 confers radioresistance of esophagus squamous cell carcinoma through regulating DNA damage response.","date":"2024","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/39672175","citation_count":2,"is_preprint":false},{"pmid":"40603489","id":"PMC_40603489","title":"Roles of MARCKSL1, MCM6, RFC4, and PLAU genes in esophageal cancer and their association with radiotherapy response.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40603489","citation_count":2,"is_preprint":false},{"pmid":"40612960","id":"PMC_40612960","title":"Comprehensive analysis of RFC4 as a potential biomarker for regulating the immune microenvironment and predicting immune therapy response in lung adenocarcinoma.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40612960","citation_count":1,"is_preprint":false},{"pmid":"40327962","id":"PMC_40327962","title":"Biallelic variants in the RFC4 gene cause a rapidly progressive congenital myopathy with severe hypotonia and axial weakness.","date":"2025","source":"Neuromuscular disorders : NMD","url":"https://pubmed.ncbi.nlm.nih.gov/40327962","citation_count":0,"is_preprint":false},{"pmid":"41872171","id":"PMC_41872171","title":"RFC4 drives temozolomide resistance in glioblastoma by activating STK38-BECN1-dependent autophagy.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41872171","citation_count":0,"is_preprint":false},{"pmid":"41574400","id":"PMC_41574400","title":"RFC4 Promotes the Metastasis of Colorectal Cancer by Regulating the Wnt/β-Catenin Pathway.","date":"2026","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/41574400","citation_count":0,"is_preprint":false},{"pmid":"42142580","id":"PMC_42142580","title":"Multidimensional Proteomics Reveals the Pro-apoptotic Mechanism of Platycodin D: Targeting RFC4 to Regulate the Notch Signaling Axis in Non-Small Cell Lung Cancer.","date":"2026","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/42142580","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10662,"output_tokens":3069,"usd":0.03901,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10606,"output_tokens":3798,"usd":0.07399,"stage2_stop_reason":"end_turn"},"total_usd":0.113,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1994,\n      \"finding\": \"RFC4 (yeast Rfc4p) encodes the 37-kDa subunit of replication factor C and is essential for yeast viability. Purified Rfc4p formed a tight complex with the Rfc3p subunit (the ATPase of RFC), establishing a direct physical interaction between these two small RFC subunits.\",\n      \"method\": \"Cloning, overexpression in E. coli, purification, biochemical characterization, protein-protein interaction assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical demonstration of Rfc4p-Rfc3p complex formation in single study; essential gene established by genetics\",\n      \"pmids\": [\"8063832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The ATP-binding domain of Rfc4 (Walker A motif, K55) is essential for DNA recognition and clamp loading by RFC. The rfc4-K55E mutant complex retained PCNA interaction and clamp loading activity but only at very high ATP concentrations, indicating Rfc4's ATPase domain contributes to ATP-dependent clamp loading.\",\n      \"method\": \"Site-directed mutagenesis of Walker A motif, overproduction of mutant RFC complexes in E. coli, in vitro ATPase assay, clamp loading assay, DNA binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple mutant complexes tested with orthogonal biochemical assays in a single rigorous study\",\n      \"pmids\": [\"11432854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Yeast Rfc4 physically interacts with the N-terminal domain of Rpa1 (Rpa1N), and this interaction is required for both DNA replication and DNA damage checkpoint function. rfc4-2 is synthetically lethal with rfa1-t11 (an Rpa1N mutation). Rfc4 functions as a sensor in the G1/S DNA damage checkpoint, intra-S checkpoint (replication block), and G2/M DNA damage checkpoint, and is epistatic with RAD24 for DNA damage sensitivity.\",\n      \"method\": \"Yeast two-hybrid/allele-specific genetic interaction (synthetic lethality screen), hydroxyurea sensitivity, DNA damage checkpoint assays (G1/S, intra-S, G2/M), epistasis analysis with rad24\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal allele-specific genetic interactions, multiple checkpoint pathway epistasis tests, and functional checkpoint readouts across multiple assays in single study\",\n      \"pmids\": [\"11340166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Drosophila Rfc4 (DmRfc4) protein localizes to all replicating nuclei but is dispersed from chromatin during mitosis. Loss-of-function mutations in DmRfc4 cause defects in the DNA replication block and DNA damage checkpoints (not the kinetochore attachment checkpoint), leading to aberrant mitotic chromosome condensation and premature sister chromatid separation.\",\n      \"method\": \"Immunofluorescence localization in larval tissue, genetic loss-of-function analysis of two alleles, checkpoint assays with DNA replication inhibitors and DNA-damaging agents\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct protein localization by immunofluorescence tied to functional consequence, two independent alleles, multiple checkpoint assays; consistent with yeast findings\",\n      \"pmids\": [\"11438670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The human RFC4 gene (encoding the p37 subunit of replication factor C) was mapped to chromosome band 3q27 by PCR from somatic hybrid DNA panels and fluorescence in situ hybridization.\",\n      \"method\": \"PCR amplification from somatic cell hybrid DNA panel, fluorescence in situ hybridization (FISH)\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct chromosomal mapping by two orthogonal methods, single study\",\n      \"pmids\": [\"7774928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RFC4 promotes nonhomologous end joining (NHEJ)-mediated DNA repair in colorectal cancer cells by physically interacting with Ku70/Ku80. RFC4 knockdown increased X-ray-induced DNA damage and apoptosis, while RFC4 did not affect homologous recombination-mediated repair.\",\n      \"method\": \"Genome-wide RNAi screen, RFC4 knockdown/overexpression, Co-immunoprecipitation with Ku70/Ku80, DNA damage assays, apoptosis assay, NHEJ and HR repair assays in vitro and xenograft mouse model\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing Ku70/Ku80 interaction, functional KD/OE with defined repair pathway readout, single lab\",\n      \"pmids\": [\"30979744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RFC4 directly binds to the Notch1 intracellular domain (NICD1) to competitively block CDK8/FBXW7-mediated ubiquitin-proteasomal degradation of NICD1, thereby stabilizing NICD1. RFC4 is itself a transcriptional target of Notch1 signaling, forming a positive feedback loop. This RFC4–NICD1 interaction promotes NSCLC metastasis and cancer stem cell properties.\",\n      \"method\": \"Co-immunoprecipitation, competitive binding assays, transcriptional reporter assays, RFC4 overexpression/knockdown, in vitro and in vivo tumor assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing direct RFC4-NICD1 interaction and functional consequences, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33976158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Bi-allelic loss-of-function variants in RFC4 that disrupt the C-terminal domain cause destabilization and reduced expression of RFC4 protein, compromised stability of other RFC complex subunits (RFC1–5), and perturbed RFC complex formation. Cell cycle studies using RFC4-deficient HeLa cells and primary fibroblasts showed perturbation of DNA replication and cell cycle progression. Structural analysis of the cryo-EM RFC-PCNA complex suggested the variants disrupt interactions within RFC4 and/or destabilize the RFC complex.\",\n      \"method\": \"Patient-derived fibroblasts and RFC4-deficient HeLa cells, Western blot for RFC complex subunit stability, RFC complex formation assay, cryo-EM structural analysis (previously determined structure), cell cycle analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cellular functional studies in patient-derived cells with multiple orthogonal methods (protein stability, complex formation, cell cycle), structural analysis; single lab\",\n      \"pmids\": [\"39106866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TMZ-induced chromatin accessibility allows transcription factor YY1 to bind the RFC4 promoter and upregulate RFC4 expression in GBM. RFC4 then stabilizes the kinase STK38, and the RFC4-STK38 interaction facilitates BECN1 recruitment, activating autophagy and conferring temozolomide resistance. Phosphorylation of STK38 at T444 stabilizes the RFC4-STK38-BECN1 complex; a T444 phospho-deficient mutant impairs autophagy.\",\n      \"method\": \"Chromatin accessibility assay, ChIP/reporter assay for YY1 binding, Co-immunoprecipitation of RFC4-STK38-BECN1, phospho-deficient mutagenesis, autophagy assays, in vivo xenograft with RFC4 overexpression and autophagy inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing RFC4-STK38-BECN1 complex, phospho-deficient mutagenesis, multiple functional assays, single lab\",\n      \"pmids\": [\"41872171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RFC4 silencing in colorectal cancer cells suppresses proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and Wnt/β-catenin signaling pathway activity. The Wnt/β-catenin pathway activator BML-284 partially reversed the inhibitory effects of RFC4 silencing on invasion and EMT, positioning RFC4 upstream of Wnt/β-catenin activation.\",\n      \"method\": \"RFC4 siRNA knockdown, proliferation/migration/invasion assays, EMT marker Western blot, Wnt/β-catenin pathway reporter/western blot, BML-284 rescue experiment, in vivo liver metastasis mouse model\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — epistasis established by pharmacological rescue, single lab, no direct binding between RFC4 and Wnt pathway components demonstrated\",\n      \"pmids\": [\"41574400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The small molecule Platycodin D (PD) binds to RFC4 (identified by thermal proteome profiling, CETSA, and PELSA), and the PD-RFC4 complex reduces nuclear entry of Notch1 and Notch3 intracellular domains, promoting their degradation via ubiquitination and downregulating Notch signaling.\",\n      \"method\": \"Thermal proteome profiling (TPP), molecular docking, CETSA, PELSA, Western blot, immunoprecipitation-Western blot (IP-WB), proteomic and ubiquitinomic profiling\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal biophysical binding assays (TPP, CETSA, PELSA) establishing direct RFC4-PD interaction, downstream signaling validated by IP-WB and proteomics; single lab\",\n      \"pmids\": [\"42142580\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RFC4 is a conserved subunit of the replication factor C (RFC) clamp loader complex whose ATP-binding domain (Walker A motif K55) is required for DNA-dependent ATPase activity and PCNA clamp loading onto DNA; it directly interacts with Rpa1N to serve as a sensor in multiple DNA damage and replication checkpoints (G1/S, intra-S, G2/M); in cancer contexts, RFC4 stabilizes NICD1 by blocking CDK8/FBXW7-mediated degradation to activate Notch signaling, promotes NHEJ-mediated DNA repair through Ku70/Ku80 interaction, and drives autophagy-based chemoresistance by stabilizing the STK38-BECN1 complex.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RFC4 is a conserved subunit of the replication factor C (RFC) clamp-loader complex that couples ATP-dependent recognition of primer-template DNA to loading of the PCNA sliding clamp during DNA replication [#0, #1]. Its ATPase domain is functionally essential: the Walker A K55 residue is required for efficient DNA-dependent clamp loading, as a K55E mutant complex loads PCNA only at very high ATP concentrations [#1]. Beyond catalysis, RFC4 physically interacts with the N-terminal domain of Rpa1 (Rpa1N), and this contact is required for both replication and the G1/S, intra-S, and G2/M DNA damage checkpoints, placing RFC4 as a sensor that is epistatic with RAD24 [#2]; consistent with this, RFC4 localizes to replicating nuclei and its loss causes checkpoint failure with aberrant chromosome condensation and premature sister chromatid separation [#3]. RFC4 is required to maintain the integrity and expression of the assembled RFC complex, and bi-allelic loss-of-function variants disrupting its C-terminal domain destabilize RFC4 and the other RFC subunits and perturb DNA replication and cell-cycle progression, defining RFC4 as the basis of a human Mendelian disorder [#7]. In cancer settings RFC4 acts in additional protein-stabilizing roles: it promotes NHEJ-mediated repair through interaction with Ku70/Ku80 [#5], directly binds NICD1 to block CDK8/FBXW7-mediated degradation and sustain Notch signaling in a positive feedback loop [#6], and stabilizes an STK38-BECN1 complex to drive autophagy-based temozolomide resistance [#8]. The small molecule Platycodin D binds RFC4 and reduces nuclear Notch intracellular domain accumulation, downregulating Notch signaling [#10].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established RFC4 as an essential structural subunit of the RFC clamp-loader and defined its first direct intra-complex contact, anchoring all later mechanism in a defined protein assembly.\",\n      \"evidence\": \"Cloning, E. coli expression, purification and interaction assay of yeast Rfc4p with Rfc3p\",\n      \"pmids\": [\"8063832\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not resolve stoichiometry or architecture of the full RFC pentamer\", \"No demonstration of clamp-loading activity for Rfc4 itself\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed that RFC4's ATPase domain is mechanistically required for ATP-dependent clamp loading, not merely structural.\",\n      \"evidence\": \"Walker A K55E site-directed mutagenesis with in vitro ATPase, DNA binding and PCNA loading assays on reconstituted RFC complexes\",\n      \"pmids\": [\"11432854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define order of ATP hydrolysis among RFC subunits\", \"No structural model of the K55 mutant in the loading reaction\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Linked RFC4 to checkpoint signaling by identifying a required Rpa1N interaction and placing RFC4 in the G1/S, intra-S, and G2/M checkpoints epistatic with RAD24.\",\n      \"evidence\": \"Allele-specific synthetic lethality (rfc4-2 / rfa1-t11), checkpoint and HU-sensitivity assays, RAD24 epistasis in yeast\",\n      \"pmids\": [\"11340166\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define the molecular signal RFC4 transmits to downstream checkpoint kinases\", \"Interaction mapped genetically/two-hybrid, not structurally\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Tied RFC4 protein localization to checkpoint function in a metazoan, showing chromatin association during replication and checkpoint-specific (not kinetochore) failures upon loss.\",\n      \"evidence\": \"Immunofluorescence and loss-of-function alleles in Drosophila with replication-block and DNA-damage checkpoint assays\",\n      \"pmids\": [\"11438670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address mammalian-specific checkpoint wiring\", \"Mechanism of chromatin dispersal at mitosis unexplained\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Mapped the human RFC4 gene to 3q27, enabling later human genetic and disease studies.\",\n      \"evidence\": \"PCR from somatic hybrid panels and FISH\",\n      \"pmids\": [\"7774928\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional information\", \"No disease association at the time\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended RFC4 function beyond replication to DNA double-strand break repair, implicating it in NHEJ through Ku70/Ku80.\",\n      \"evidence\": \"Genome-wide RNAi screen, Co-IP with Ku70/Ku80, NHEJ vs HR repair and apoptosis assays in colorectal cancer cells and xenografts\",\n      \"pmids\": [\"30979744\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab Co-IP without reciprocal/structural validation\", \"Whether NHEJ role depends on intact RFC complex unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a non-replicative protein-stabilization role: RFC4 directly binds NICD1 to block its degradation, sustaining Notch signaling in a feedback loop driving metastasis.\",\n      \"evidence\": \"Co-IP, competitive binding and reporter assays, knockdown/overexpression, in vitro and in vivo NSCLC tumor assays\",\n      \"pmids\": [\"33976158\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface on RFC4 not mapped\", \"Whether this requires monomeric RFC4 or the RFC complex is unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established RFC4 as the cause of a human Mendelian disorder by showing C-terminal loss-of-function variants destabilize RFC4 and the whole RFC complex with replication/cell-cycle defects.\",\n      \"evidence\": \"Patient fibroblasts and RFC4-deficient HeLa cells, Western blot of RFC subunit stability, complex-formation and cell-cycle assays, cryo-EM structural interpretation\",\n      \"pmids\": [\"39106866\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genotype-phenotype spectrum not fully defined\", \"Direct structural data on patient variants not generated\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed RFC4 is transcriptionally inducible (via YY1) and stabilizes an STK38-BECN1 complex to activate autophagy and confer chemoresistance.\",\n      \"evidence\": \"Chromatin accessibility/ChIP-reporter for YY1, Co-IP of RFC4-STK38-BECN1, T444 phospho-deficient mutagenesis, autophagy assays and xenografts in GBM\",\n      \"pmids\": [\"41872171\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct RFC4-STK38 binding interface unmapped\", \"Single lab; relationship to RFC4's clamp-loader role unaddressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified RFC4 as a druggable target whose engagement by Platycodin D suppresses nuclear Notch intracellular domain accumulation and Notch signaling.\",\n      \"evidence\": \"Thermal proteome profiling, CETSA, PELSA binding assays, IP-WB, proteomic and ubiquitinomic profiling\",\n      \"pmids\": [\"42142580\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site of Platycodin D on RFC4 not defined\", \"Mechanistic link between PD binding and Notch nuclear exclusion not fully resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Positioned RFC4 upstream of Wnt/beta-catenin-driven invasion and EMT in colorectal cancer.\",\n      \"evidence\": \"siRNA knockdown with proliferation/invasion/EMT assays and pharmacological BML-284 rescue, liver metastasis model\",\n      \"pmids\": [\"41574400\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct binding between RFC4 and Wnt pathway components demonstrated\", \"Epistasis rests on pharmacological rescue alone\", \"Single lab, not independently confirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RFC4's canonical clamp-loader/checkpoint functions mechanistically relate to its many cancer-associated protein-stabilization roles (NICD1, Ku70/80, STK38-BECN1, Wnt) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Unclear whether non-replicative roles require the intact RFC complex or free RFC4\", \"No shared structural binding interface identified across partners\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"Replication factor C (RFC) clamp-loader complex\"],\n    \"partners\": [\"RFC3\", \"RPA1\", \"KU70\", \"KU80\", \"NICD1\", \"STK38\", \"BECN1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}