{"gene":"RFC5","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1996,"finding":"Rfc5 is a small subunit of the replication factor C (RFC) complex in S. cerevisiae and is required for the S-phase checkpoint that couples DNA replication to mitotic entry. The rfc5-1 temperature-sensitive mutation causes cells to enter mitosis with unevenly separated or fragmented chromosomes, and overexpression of SPK1/Rad53 suppresses this defect, placing Rfc5 upstream of Rad53 in the checkpoint pathway. Overexpression of PCNA (POL30) suppresses the replication defect but not the checkpoint defect, indicating RFC has a direct role in sensing replication state and transmitting the checkpoint signal.","method":"Genetic epistasis (suppressor screens, temperature-sensitive mutant analysis, overexpression of SPK1/POL30), cell biology (chromosome segregation analysis)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple suppressor relationships and orthogonal readouts (replication vs. checkpoint phenotype dissection), replicated across multiple subsequent studies","pmids":["8692942"],"is_preprint":false},{"year":1997,"finding":"Rfc5 is required for the DNA damage checkpoint: in rfc5-1 mutants, Rad53 phosphorylation in response to DNA damage is reduced during S phase, RNR3 transcription induction is impaired, and S-phase progression is not slowed in response to DNA damage. Overexpression of TEL1 suppresses rfc5-1 defects and restores Rad53 phosphorylation and RNR3 induction, placing Rfc5 in the Mec1/Tel1-Rad53 signaling axis upstream of Rad53 activation.","method":"Genetic epistasis (suppressor analysis with TEL1, RAD53 overexpression), phosphorylation assays (Rad53 phosphorylation shift), transcription induction assay (RNR3)","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal readouts (Rad53 phosphorylation, RNR3 induction, S-phase progression, suppressor genetics), consistent with and replicated by subsequent studies","pmids":["9315648"],"is_preprint":false},{"year":1998,"finding":"Rad24, a protein structurally related to RFC subunits, physically interacts with RFC subunits Rfc2 and Rfc5 and co-sediments with Rfc5. RAD24 overexpression suppresses rfc5-1 sensitivity to DNA-damaging agents and restores Rad53 phosphorylation, demonstrating a physical and functional interaction between Rad24 and Rfc5 in checkpoint pathways.","method":"Co-immunoprecipitation, co-sedimentation, genetic suppressor analysis, Rad53 phosphorylation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal physical interaction (co-IP and co-sedimentation) combined with functional genetic epistasis and biochemical readout (Rad53 phosphorylation)","pmids":["9710632"],"is_preprint":false},{"year":2000,"finding":"RFC5 functions in G1-, S-, and G2/M-phase DNA damage checkpoints in cooperation with Rad24. In rfc5-1 rad24-K115R double mutants, G1 and G2/M checkpoint defects appear that are absent in either single mutant. Co-immunoprecipitation showed that the Rad24(K115R) protein (NTP-binding motif mutant) fails to interact with RFC proteins in rfc5-1 mutants, establishing that the Rad24–RFC interaction is essential for checkpoint control across all cell cycle phases.","method":"Co-immunoprecipitation, site-directed mutagenesis of NTP-binding motif (K115E/K115R), double-mutant genetic epistasis, cell cycle checkpoint assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis of conserved motif combined with co-IP and genetic double-mutant analysis, multiple orthogonal methods","pmids":["10913172"],"is_preprint":false},{"year":2000,"finding":"RFC5 suppressor mutations that rescue rfc1-1 cold-sensitive growth map to conserved RFC box motifs IV–VII of Rfc5p, regions predicted to mediate inter-subunit contacts. These RFC5 suppressors do not interfere with Rad53 phosphorylation, unlike the checkpoint-defective rfc5-1 mutation, separating replication and checkpoint functions and suggesting RFC box motifs IV–VII coordinate neighboring-subunit interactions within the RFC complex.","method":"Genetic suppressor screen (isolation and characterization of RFC5 suppressor alleles), phenotypic analysis (MMS/HU sensitivity, telomere length, mutator phenotype), Rad53 phosphorylation assay, structural comparison","journal":"Molecular & general genetics : MGG","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — suppressor genetics with multiple phenotypic readouts and biochemical (Rad53 phosphorylation) validation; single lab, structural inference is comparative not direct","pmids":["11129041"],"is_preprint":false},{"year":1995,"finding":"Human RFC5 (p36.5 subunit) is a component of the multimeric human replication factor C complex, which is essential for processive DNA chain elongation by DNA polymerase delta or epsilon. The RFC5 gene was mapped to human chromosome band 12q24.2–q24.3.","method":"PCR amplification from somatic hybrid DNAs, fluorescence in situ hybridization (FISH)","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — chromosomal mapping establishes human RFC5 as a genuine RFC complex component, but no direct functional assay of Rfc5 activity was performed","pmids":["7774928"],"is_preprint":false},{"year":2017,"finding":"FoxM1 transcriptionally activates RFC5 expression by directly interacting with the RFC5 promoter. Knockdown of FoxM1 or RFC5 re-sensitizes glioma cells to temozolomide, indicating the FoxM1–RFC5 transcriptional axis mediates TMZ resistance.","method":"Promoter interaction assay (FoxM1 binding to RFC5 promoter), siRNA knockdown of FoxM1/RFC5, cell viability/apoptosis assays, pharmacological inhibition (thiostrepton)","journal":"Cell biology and toxicology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — promoter interaction and knockdown phenotype with two-target validation; single lab, no ChIP or reconstitution","pmids":["28185110"],"is_preprint":false},{"year":2021,"finding":"AEG-1 positively regulates RFC5 expression in glioma cells (identified by gene expression array). AEG-1 knockdown reduces RFC5 levels and impairs homologous recombination DNA repair activity induced by ionizing radiation, demonstrating that RFC5 is required for HR repair in glioma cells.","method":"Affymetrix gene expression array (AEG-1 KD → RFC5 downregulation), γ-H2AX foci assay, colony formation assay, flow cytometry, HR repair activity assay","journal":"DNA and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — HR repair activity assay with γ-H2AX readout; AEG-1→RFC5 link is correlative from array, single lab","pmids":["34042508"],"is_preprint":false},{"year":2025,"finding":"High RFC5 expression in nasopharyngeal carcinoma cells enhances cisplatin-induced DNA damage repair, reduces micronucleus formation, suppresses cGAS-STING pathway activation, limits inflammatory mediator production, and promotes T cell exhaustion (elevated PD-1, LAG-3, CTLA-4; reduced IFN-γ and TNF-α secretion by CD8+ T cells) in vivo, linking RFC5-mediated DNA repair to immune evasion.","method":"RFC5 expression manipulation in NPC cells, micronuclei quantification, cGAS-STING pathway activity assays, in vivo tumor immune microenvironment analysis (cytokine measurement, T cell marker expression)","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple mechanistic readouts (micronuclei, cGAS-STING, immune markers) in a single study; single lab, no reconstitution or direct RFC5 biochemical assay","pmids":["41192524"],"is_preprint":false},{"year":2024,"finding":"SRSF10 promotes colorectal cancer progression by generating an aberrantly spliced exclusion isoform of RFC5 (AS1 exclusion of exon 2). SRSF10 knockdown alters RFC5 pre-mRNA splicing (detected by agarose gel electrophoresis of transcripts), affecting DNA replication and cell cycle progression in CRC cells.","method":"SRSF10 knockdown/overexpression, agarose gel electrophoresis of RFC5 splice variants, CCK8, transwell, flow cytometry","journal":"Technology in cancer research & treatment","confidence":"Low","confidence_rationale":"Tier 3 / Weak — splicing change detected by gel electrophoresis, single lab, no direct functional validation of the specific RFC5 isoform","pmids":["39110418"],"is_preprint":false}],"current_model":"RFC5 encodes a small subunit of the replication factor C (RFC) clamp-loader complex that is essential for processive DNA replication by DNA polymerase delta/epsilon; beyond replication, Rfc5 acts as a sensor/transducer in DNA damage and replication-block checkpoints by physically interacting with Rad24 (via conserved RFC box motifs) and facilitating Rad53 (Mec1/Tel1 pathway) phosphorylation across all cell cycle phases, while in human cancer cells RFC5 additionally supports homologous recombination repair, suppresses cGAS-STING-mediated innate immune signaling, and is transcriptionally regulated by FoxM1 and post-transcriptionally regulated by alternative splicing via SRSF10."},"narrative":{"mechanistic_narrative":"RFC5 encodes a small subunit of the replication factor C (RFC) clamp-loader complex, which is essential for processive DNA chain elongation by DNA polymerase delta/epsilon [PMID:7774928, PMID:8692942]. Beyond its replication role, Rfc5 acts as a sensor and transducer of replication and DNA-damage checkpoint signals: the rfc5-1 mutation abolishes the S-phase checkpoint and impairs DNA-damage-induced Rad53 phosphorylation and RNR3 induction, defects rescued by overexpression of SPK1/Rad53 or TEL1, placing Rfc5 upstream of Rad53 in the Mec1/Tel1 signaling axis [PMID:8692942, PMID:9315648]. Rfc5 physically interacts with the RFC-related checkpoint protein Rad24, an interaction dependent on Rad24's NTP-binding motif and required for checkpoint control across G1, S, and G2/M phases [PMID:9710632, PMID:10913172]. RFC box motifs IV–VII of Rfc5 mediate inter-subunit contacts within the complex, and the replication and checkpoint functions are genetically separable [PMID:11129041]. In human cancer cells, RFC5 supports homologous-recombination DNA repair and, by enhancing repair of chemotherapy-induced lesions, suppresses cGAS-STING innate-immune signaling to promote immune evasion [PMID:34042508, PMID:41192524]; its expression is transcriptionally activated by FoxM1, contributing to chemoresistance [PMID:28185110].","teleology":[{"year":1995,"claim":"Established the human gene product as a bona fide subunit of the multimeric RFC clamp loader required for processive DNA polymerase delta/epsilon activity, anchoring RFC5 to the replication machinery.","evidence":"PCR from somatic hybrid DNAs and FISH mapping of human RFC5 (p36.5)","pmids":["7774928"],"confidence":"Medium","gaps":["No direct biochemical assay of Rfc5 clamp-loading activity","Subunit stoichiometry and contacts not resolved"]},{"year":1996,"claim":"Showed RFC has a function beyond replication itself — sensing replication state and transmitting an S-phase checkpoint signal — by separating the replication defect (PCNA-suppressible) from the checkpoint defect (Rad53-suppressible).","evidence":"Temperature-sensitive rfc5-1 mutant analysis and SPK1/POL30 overexpression suppression in S. cerevisiae, with chromosome segregation readout","pmids":["8692942"],"confidence":"High","gaps":["Molecular nature of the replication-state signal sensed by RFC unknown","Direct biochemical link to Rad53 not shown"]},{"year":1997,"claim":"Placed Rfc5 in the Mec1/Tel1-Rad53 DNA-damage checkpoint axis, demonstrating it is needed to activate Rad53 and downstream transcriptional responses after damage during S phase.","evidence":"Rad53 phosphorylation shift, RNR3 induction, S-phase progression assays, and TEL1/RAD53 suppressor genetics in rfc5-1 mutants","pmids":["9315648"],"confidence":"High","gaps":["Whether Rfc5 directly contacts Mec1/Tel1 not established","Mechanism of signal relay to Rad53 unresolved"]},{"year":1998,"claim":"Identified a physical partner for the checkpoint function — the RFC-related protein Rad24 — providing a molecular basis for how RFC subunits couple to the damage-response apparatus.","evidence":"Co-immunoprecipitation, co-sedimentation, and RAD24-overexpression suppression of rfc5-1 with Rad53 phosphorylation readout","pmids":["9710632"],"confidence":"High","gaps":["Whether Rad24-Rfc5 forms a distinct alternative clamp-loader not defined here","Direct binding interface not mapped"]},{"year":2000,"claim":"Demonstrated the Rad24–RFC interaction operates across all cell cycle phases and depends on Rad24's NTP-binding motif, generalizing the checkpoint role beyond S phase.","evidence":"Co-IP with Rad24 K115R/K115E motif mutants and rfc5-1 rad24 double-mutant checkpoint assays in G1, S, G2/M","pmids":["10913172"],"confidence":"High","gaps":["Structural basis of NTP-dependent interaction not resolved","Loaded sensor structure unknown"]},{"year":2000,"claim":"Genetically separated RFC5's replication and checkpoint functions and mapped inter-subunit contact determinants to conserved RFC box motifs IV–VII.","evidence":"RFC5 suppressor screen of rfc1-1, phenotypic readouts (MMS/HU sensitivity, telomere length, mutator), Rad53 phosphorylation, comparative structural inference","pmids":["11129041"],"confidence":"Medium","gaps":["Structural inference is comparative, not direct","Single-lab characterization"]},{"year":2017,"claim":"Connected RFC5 to chemoresistance in human cancer by identifying transcriptional control through FoxM1.","evidence":"FoxM1 promoter-interaction assay, siRNA knockdown of FoxM1/RFC5, viability/apoptosis assays and thiostrepton inhibition in glioma cells","pmids":["28185110"],"confidence":"Medium","gaps":["No ChIP confirmation of direct promoter binding","Single lab, no reconstitution"]},{"year":2021,"claim":"Implicated RFC5 in homologous-recombination repair in human cells, extending its DNA-metabolism role beyond replication.","evidence":"AEG-1 knockdown gene-expression array, γ-H2AX foci, colony formation, HR repair activity assay after ionizing radiation in glioma","pmids":["34042508"],"confidence":"Medium","gaps":["AEG-1→RFC5 link is correlative from array","Direct role of RFC5 in HR step not mechanistically dissected"]},{"year":2025,"claim":"Linked RFC5-mediated DNA repair to tumor immune evasion, showing repair of chemotherapy lesions suppresses cytosolic DNA sensing and dampens anti-tumor immunity.","evidence":"RFC5 manipulation in NPC cells, micronuclei quantification, cGAS-STING assays, in vivo cytokine and T-cell exhaustion marker analysis","pmids":["41192524"],"confidence":"Medium","gaps":["No direct RFC5 biochemical assay","Single study, single lab","Causal chain from repair to STING suppression inferred"]},{"year":2024,"claim":"Raised the possibility that RFC5 function is modulated post-transcriptionally through SRSF10-dependent alternative splicing.","evidence":"SRSF10 knockdown/overexpression with gel electrophoresis of RFC5 splice variants and proliferation/cell-cycle assays in colorectal cancer cells","pmids":["39110418"],"confidence":"Low","gaps":["Splicing change detected only by gel electrophoresis","Functional consequence of the specific exon-2-excluded isoform not validated","Single lab"]},{"year":null,"claim":"How RFC5 mechanistically transduces a replication/damage signal to Rad53/Mec1 at the molecular level, and whether the human clamp loader retains a distinct checkpoint-sensing role, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of an RFC5-containing checkpoint sensor","Direct enzymatic activity of human RFC5 within HR not defined","Mechanism linking repair to cGAS-STING suppression not biochemically established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[5,0]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,2]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[5,0]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,3]}],"complexes":["Replication factor C (RFC) complex"],"partners":["RAD24","RFC2","RAD53"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P40937","full_name":"Replication factor C subunit 5","aliases":["Activator 1 36 kDa subunit","A1 36 kDa subunit","Activator 1 subunit 5","Replication factor C 36 kDa subunit","RF-C 36 kDa subunit","RFC36"],"length_aa":340,"mass_kda":38.5,"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","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P40937/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RFC5","classification":"Common Essential","n_dependent_lines":1187,"n_total_lines":1208,"dependency_fraction":0.9826158940397351},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DRG1","stoichiometry":0.2},{"gene":"H2AFZ","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"HMGA1","stoichiometry":0.2},{"gene":"HUS1","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2},{"gene":"NUCKS1","stoichiometry":0.2},{"gene":"NUMA1","stoichiometry":0.2},{"gene":"PARP1","stoichiometry":0.2},{"gene":"RACK1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RFC5","total_profiled":1310},"omim":[{"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":"600407","title":"REPLICATION FACTOR C, SUBUNIT 5; RFC5","url":"https://www.omim.org/entry/600407"},{"mim_id":"600405","title":"REPLICATION FACTOR C, SUBUNIT 3; RFC3","url":"https://www.omim.org/entry/600405"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RFC5"},"hgnc":{"alias_symbol":["RFC36"],"prev_symbol":[]},"alphafold":{"accession":"P40937","domains":[{"cath_id":"3.40.50.300","chopping":"19-174","consensus_level":"high","plddt":91.5181,"start":19,"end":174},{"cath_id":"1.10.8.60","chopping":"180-240","consensus_level":"high","plddt":96.1731,"start":180,"end":240},{"cath_id":"1.20.272.10","chopping":"245-340","consensus_level":"high","plddt":93.8001,"start":245,"end":340}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P40937","model_url":"https://alphafold.ebi.ac.uk/files/AF-P40937-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P40937-F1-predicted_aligned_error_v6.png","plddt_mean":90.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RFC5","jax_strain_url":"https://www.jax.org/strain/search?query=RFC5"},"sequence":{"accession":"P40937","fasta_url":"https://rest.uniprot.org/uniprotkb/P40937.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P40937/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P40937"}},"corpus_meta":[{"pmid":"8692942","id":"PMC_8692942","title":"Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8692942","citation_count":87,"is_preprint":false},{"pmid":"9315648","id":"PMC_9315648","title":"Rfc5, a replication factor C component, is required for regulation of Rad53 protein kinase in the yeast checkpoint pathway.","date":"1997","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9315648","citation_count":80,"is_preprint":false},{"pmid":"9710632","id":"PMC_9710632","title":"Functional and physical interaction between Rad24 and Rfc5 in the yeast checkpoint pathways.","date":"1998","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9710632","citation_count":69,"is_preprint":false},{"pmid":"10913172","id":"PMC_10913172","title":"Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae.","date":"2000","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10913172","citation_count":67,"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":"28185110","id":"PMC_28185110","title":"FoxM1-mediated RFC5 expression promotes temozolomide resistance.","date":"2017","source":"Cell biology and toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/28185110","citation_count":37,"is_preprint":false},{"pmid":"34042508","id":"PMC_34042508","title":"AEG-1 Knockdown Sensitizes Glioma Cells to Radiation Through Impairing Homologous Recombination Via Targeting RFC5.","date":"2021","source":"DNA and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/34042508","citation_count":11,"is_preprint":false},{"pmid":"36988339","id":"PMC_36988339","title":"RFC5, regulated by circ_0038985/miR-3614-5p, functions as an oncogene in the progression of colorectal cancer.","date":"2023","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/36988339","citation_count":10,"is_preprint":false},{"pmid":"39110418","id":"PMC_39110418","title":"High Expression of SRSF10 Promotes Colorectal Cancer Progression by Aberrant Alternative Splicing of RFC5.","date":"2024","source":"Technology in cancer research & treatment","url":"https://pubmed.ncbi.nlm.nih.gov/39110418","citation_count":6,"is_preprint":false},{"pmid":"11129041","id":"PMC_11129041","title":"Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions.","date":"2000","source":"Molecular & general genetics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/11129041","citation_count":5,"is_preprint":false},{"pmid":"41192524","id":"PMC_41192524","title":"RFC5 enhances DNA damage response and immune escape via suppressing the cGAS-STING pathway in nasopharyngeal carcinoma.","date":"2025","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/41192524","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6949,"output_tokens":2896,"usd":0.032143,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10358,"output_tokens":3351,"usd":0.067782,"stage2_stop_reason":"end_turn"},"total_usd":0.099925,"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\": 1996,\n      \"finding\": \"Rfc5 is a small subunit of the replication factor C (RFC) complex in S. cerevisiae and is required for the S-phase checkpoint that couples DNA replication to mitotic entry. The rfc5-1 temperature-sensitive mutation causes cells to enter mitosis with unevenly separated or fragmented chromosomes, and overexpression of SPK1/Rad53 suppresses this defect, placing Rfc5 upstream of Rad53 in the checkpoint pathway. Overexpression of PCNA (POL30) suppresses the replication defect but not the checkpoint defect, indicating RFC has a direct role in sensing replication state and transmitting the checkpoint signal.\",\n      \"method\": \"Genetic epistasis (suppressor screens, temperature-sensitive mutant analysis, overexpression of SPK1/POL30), cell biology (chromosome segregation analysis)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple suppressor relationships and orthogonal readouts (replication vs. checkpoint phenotype dissection), replicated across multiple subsequent studies\",\n      \"pmids\": [\"8692942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Rfc5 is required for the DNA damage checkpoint: in rfc5-1 mutants, Rad53 phosphorylation in response to DNA damage is reduced during S phase, RNR3 transcription induction is impaired, and S-phase progression is not slowed in response to DNA damage. Overexpression of TEL1 suppresses rfc5-1 defects and restores Rad53 phosphorylation and RNR3 induction, placing Rfc5 in the Mec1/Tel1-Rad53 signaling axis upstream of Rad53 activation.\",\n      \"method\": \"Genetic epistasis (suppressor analysis with TEL1, RAD53 overexpression), phosphorylation assays (Rad53 phosphorylation shift), transcription induction assay (RNR3)\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal readouts (Rad53 phosphorylation, RNR3 induction, S-phase progression, suppressor genetics), consistent with and replicated by subsequent studies\",\n      \"pmids\": [\"9315648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Rad24, a protein structurally related to RFC subunits, physically interacts with RFC subunits Rfc2 and Rfc5 and co-sediments with Rfc5. RAD24 overexpression suppresses rfc5-1 sensitivity to DNA-damaging agents and restores Rad53 phosphorylation, demonstrating a physical and functional interaction between Rad24 and Rfc5 in checkpoint pathways.\",\n      \"method\": \"Co-immunoprecipitation, co-sedimentation, genetic suppressor analysis, Rad53 phosphorylation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal physical interaction (co-IP and co-sedimentation) combined with functional genetic epistasis and biochemical readout (Rad53 phosphorylation)\",\n      \"pmids\": [\"9710632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"RFC5 functions in G1-, S-, and G2/M-phase DNA damage checkpoints in cooperation with Rad24. In rfc5-1 rad24-K115R double mutants, G1 and G2/M checkpoint defects appear that are absent in either single mutant. Co-immunoprecipitation showed that the Rad24(K115R) protein (NTP-binding motif mutant) fails to interact with RFC proteins in rfc5-1 mutants, establishing that the Rad24–RFC interaction is essential for checkpoint control across all cell cycle phases.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis of NTP-binding motif (K115E/K115R), double-mutant genetic epistasis, cell cycle checkpoint assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis of conserved motif combined with co-IP and genetic double-mutant analysis, multiple orthogonal methods\",\n      \"pmids\": [\"10913172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"RFC5 suppressor mutations that rescue rfc1-1 cold-sensitive growth map to conserved RFC box motifs IV–VII of Rfc5p, regions predicted to mediate inter-subunit contacts. These RFC5 suppressors do not interfere with Rad53 phosphorylation, unlike the checkpoint-defective rfc5-1 mutation, separating replication and checkpoint functions and suggesting RFC box motifs IV–VII coordinate neighboring-subunit interactions within the RFC complex.\",\n      \"method\": \"Genetic suppressor screen (isolation and characterization of RFC5 suppressor alleles), phenotypic analysis (MMS/HU sensitivity, telomere length, mutator phenotype), Rad53 phosphorylation assay, structural comparison\",\n      \"journal\": \"Molecular & general genetics : MGG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — suppressor genetics with multiple phenotypic readouts and biochemical (Rad53 phosphorylation) validation; single lab, structural inference is comparative not direct\",\n      \"pmids\": [\"11129041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Human RFC5 (p36.5 subunit) is a component of the multimeric human replication factor C complex, which is essential for processive DNA chain elongation by DNA polymerase delta or epsilon. The RFC5 gene was mapped to human chromosome band 12q24.2–q24.3.\",\n      \"method\": \"PCR amplification from somatic hybrid DNAs, fluorescence in situ hybridization (FISH)\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — chromosomal mapping establishes human RFC5 as a genuine RFC complex component, but no direct functional assay of Rfc5 activity was performed\",\n      \"pmids\": [\"7774928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FoxM1 transcriptionally activates RFC5 expression by directly interacting with the RFC5 promoter. Knockdown of FoxM1 or RFC5 re-sensitizes glioma cells to temozolomide, indicating the FoxM1–RFC5 transcriptional axis mediates TMZ resistance.\",\n      \"method\": \"Promoter interaction assay (FoxM1 binding to RFC5 promoter), siRNA knockdown of FoxM1/RFC5, cell viability/apoptosis assays, pharmacological inhibition (thiostrepton)\",\n      \"journal\": \"Cell biology and toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — promoter interaction and knockdown phenotype with two-target validation; single lab, no ChIP or reconstitution\",\n      \"pmids\": [\"28185110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AEG-1 positively regulates RFC5 expression in glioma cells (identified by gene expression array). AEG-1 knockdown reduces RFC5 levels and impairs homologous recombination DNA repair activity induced by ionizing radiation, demonstrating that RFC5 is required for HR repair in glioma cells.\",\n      \"method\": \"Affymetrix gene expression array (AEG-1 KD → RFC5 downregulation), γ-H2AX foci assay, colony formation assay, flow cytometry, HR repair activity assay\",\n      \"journal\": \"DNA and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — HR repair activity assay with γ-H2AX readout; AEG-1→RFC5 link is correlative from array, single lab\",\n      \"pmids\": [\"34042508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"High RFC5 expression in nasopharyngeal carcinoma cells enhances cisplatin-induced DNA damage repair, reduces micronucleus formation, suppresses cGAS-STING pathway activation, limits inflammatory mediator production, and promotes T cell exhaustion (elevated PD-1, LAG-3, CTLA-4; reduced IFN-γ and TNF-α secretion by CD8+ T cells) in vivo, linking RFC5-mediated DNA repair to immune evasion.\",\n      \"method\": \"RFC5 expression manipulation in NPC cells, micronuclei quantification, cGAS-STING pathway activity assays, in vivo tumor immune microenvironment analysis (cytokine measurement, T cell marker expression)\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple mechanistic readouts (micronuclei, cGAS-STING, immune markers) in a single study; single lab, no reconstitution or direct RFC5 biochemical assay\",\n      \"pmids\": [\"41192524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SRSF10 promotes colorectal cancer progression by generating an aberrantly spliced exclusion isoform of RFC5 (AS1 exclusion of exon 2). SRSF10 knockdown alters RFC5 pre-mRNA splicing (detected by agarose gel electrophoresis of transcripts), affecting DNA replication and cell cycle progression in CRC cells.\",\n      \"method\": \"SRSF10 knockdown/overexpression, agarose gel electrophoresis of RFC5 splice variants, CCK8, transwell, flow cytometry\",\n      \"journal\": \"Technology in cancer research & treatment\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — splicing change detected by gel electrophoresis, single lab, no direct functional validation of the specific RFC5 isoform\",\n      \"pmids\": [\"39110418\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RFC5 encodes a small subunit of the replication factor C (RFC) clamp-loader complex that is essential for processive DNA replication by DNA polymerase delta/epsilon; beyond replication, Rfc5 acts as a sensor/transducer in DNA damage and replication-block checkpoints by physically interacting with Rad24 (via conserved RFC box motifs) and facilitating Rad53 (Mec1/Tel1 pathway) phosphorylation across all cell cycle phases, while in human cancer cells RFC5 additionally supports homologous recombination repair, suppresses cGAS-STING-mediated innate immune signaling, and is transcriptionally regulated by FoxM1 and post-transcriptionally regulated by alternative splicing via SRSF10.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RFC5 encodes a small subunit of the replication factor C (RFC) clamp-loader complex, which is essential for processive DNA chain elongation by DNA polymerase delta/epsilon [#5, #0]. Beyond its replication role, Rfc5 acts as a sensor and transducer of replication and DNA-damage checkpoint signals: the rfc5-1 mutation abolishes the S-phase checkpoint and impairs DNA-damage-induced Rad53 phosphorylation and RNR3 induction, defects rescued by overexpression of SPK1/Rad53 or TEL1, placing Rfc5 upstream of Rad53 in the Mec1/Tel1 signaling axis [#0, #1]. Rfc5 physically interacts with the RFC-related checkpoint protein Rad24, an interaction dependent on Rad24's NTP-binding motif and required for checkpoint control across G1, S, and G2/M phases [#2, #3]. RFC box motifs IV–VII of Rfc5 mediate inter-subunit contacts within the complex, and the replication and checkpoint functions are genetically separable [#4]. In human cancer cells, RFC5 supports homologous-recombination DNA repair and, by enhancing repair of chemotherapy-induced lesions, suppresses cGAS-STING innate-immune signaling to promote immune evasion [#7, #8]; its expression is transcriptionally activated by FoxM1, contributing to chemoresistance [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established the human gene product as a bona fide subunit of the multimeric RFC clamp loader required for processive DNA polymerase delta/epsilon activity, anchoring RFC5 to the replication machinery.\",\n      \"evidence\": \"PCR from somatic hybrid DNAs and FISH mapping of human RFC5 (p36.5)\",\n      \"pmids\": [\"7774928\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct biochemical assay of Rfc5 clamp-loading activity\", \"Subunit stoichiometry and contacts not resolved\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showed RFC has a function beyond replication itself — sensing replication state and transmitting an S-phase checkpoint signal — by separating the replication defect (PCNA-suppressible) from the checkpoint defect (Rad53-suppressible).\",\n      \"evidence\": \"Temperature-sensitive rfc5-1 mutant analysis and SPK1/POL30 overexpression suppression in S. cerevisiae, with chromosome segregation readout\",\n      \"pmids\": [\"8692942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular nature of the replication-state signal sensed by RFC unknown\", \"Direct biochemical link to Rad53 not shown\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Placed Rfc5 in the Mec1/Tel1-Rad53 DNA-damage checkpoint axis, demonstrating it is needed to activate Rad53 and downstream transcriptional responses after damage during S phase.\",\n      \"evidence\": \"Rad53 phosphorylation shift, RNR3 induction, S-phase progression assays, and TEL1/RAD53 suppressor genetics in rfc5-1 mutants\",\n      \"pmids\": [\"9315648\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Rfc5 directly contacts Mec1/Tel1 not established\", \"Mechanism of signal relay to Rad53 unresolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identified a physical partner for the checkpoint function — the RFC-related protein Rad24 — providing a molecular basis for how RFC subunits couple to the damage-response apparatus.\",\n      \"evidence\": \"Co-immunoprecipitation, co-sedimentation, and RAD24-overexpression suppression of rfc5-1 with Rad53 phosphorylation readout\",\n      \"pmids\": [\"9710632\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Rad24-Rfc5 forms a distinct alternative clamp-loader not defined here\", \"Direct binding interface not mapped\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrated the Rad24–RFC interaction operates across all cell cycle phases and depends on Rad24's NTP-binding motif, generalizing the checkpoint role beyond S phase.\",\n      \"evidence\": \"Co-IP with Rad24 K115R/K115E motif mutants and rfc5-1 rad24 double-mutant checkpoint assays in G1, S, G2/M\",\n      \"pmids\": [\"10913172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NTP-dependent interaction not resolved\", \"Loaded sensor structure unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Genetically separated RFC5's replication and checkpoint functions and mapped inter-subunit contact determinants to conserved RFC box motifs IV–VII.\",\n      \"evidence\": \"RFC5 suppressor screen of rfc1-1, phenotypic readouts (MMS/HU sensitivity, telomere length, mutator), Rad53 phosphorylation, comparative structural inference\",\n      \"pmids\": [\"11129041\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural inference is comparative, not direct\", \"Single-lab characterization\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected RFC5 to chemoresistance in human cancer by identifying transcriptional control through FoxM1.\",\n      \"evidence\": \"FoxM1 promoter-interaction assay, siRNA knockdown of FoxM1/RFC5, viability/apoptosis assays and thiostrepton inhibition in glioma cells\",\n      \"pmids\": [\"28185110\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ChIP confirmation of direct promoter binding\", \"Single lab, no reconstitution\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated RFC5 in homologous-recombination repair in human cells, extending its DNA-metabolism role beyond replication.\",\n      \"evidence\": \"AEG-1 knockdown gene-expression array, γ-H2AX foci, colony formation, HR repair activity assay after ionizing radiation in glioma\",\n      \"pmids\": [\"34042508\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"AEG-1→RFC5 link is correlative from array\", \"Direct role of RFC5 in HR step not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked RFC5-mediated DNA repair to tumor immune evasion, showing repair of chemotherapy lesions suppresses cytosolic DNA sensing and dampens anti-tumor immunity.\",\n      \"evidence\": \"RFC5 manipulation in NPC cells, micronuclei quantification, cGAS-STING assays, in vivo cytokine and T-cell exhaustion marker analysis\",\n      \"pmids\": [\"41192524\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct RFC5 biochemical assay\", \"Single study, single lab\", \"Causal chain from repair to STING suppression inferred\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Raised the possibility that RFC5 function is modulated post-transcriptionally through SRSF10-dependent alternative splicing.\",\n      \"evidence\": \"SRSF10 knockdown/overexpression with gel electrophoresis of RFC5 splice variants and proliferation/cell-cycle assays in colorectal cancer cells\",\n      \"pmids\": [\"39110418\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Splicing change detected only by gel electrophoresis\", \"Functional consequence of the specific exon-2-excluded isoform not validated\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RFC5 mechanistically transduces a replication/damage signal to Rad53/Mec1 at the molecular level, and whether the human clamp loader retains a distinct checkpoint-sensing role, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of an RFC5-containing checkpoint sensor\", \"Direct enzymatic activity of human RFC5 within HR not defined\", \"Mechanism linking repair to cGAS-STING suppression not biochemically established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [5, 0]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [5, 0]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"complexes\": [\"Replication factor C (RFC) complex\"],\n    \"partners\": [\"RAD24\", \"RFC2\", \"RAD53\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}