{"gene":"RFWD3","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2010,"finding":"RFWD3 forms a complex with Mdm2 and p53, synergistically ubiquitinates p53 in vitro, and is required to stabilize p53 in the late response to DNA damage. RFWD3-Mdm2 complex restricts polyubiquitination of p53 by Mdm2 alone. RFWD3 is phosphorylated by ATM/ATR kinases, and phosphorylation mutants fail to stimulate p53 ubiquitination.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, phosphorylation-deficient mutant analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 1-2 / Weak — in vitro ubiquitination assay and Co-IP from a single lab with two orthogonal methods","pmids":["20173098"],"is_preprint":false},{"year":2011,"finding":"RFWD3 directly interacts with RPA (specifically RPA2 subunit) and is recruited to sites of DNA damage in a RPA-dependent manner. The C-terminus of RFWD3 (coiled-coil and WD40 domains) is necessary for RPA binding. Loss of RFWD3 results in persistent γH2AX and RAD51 foci in damaged cells.","method":"Co-immunoprecipitation, pulldown with purified proteins, immunofluorescence, deletion mutant analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction confirmed with purified proteins and reciprocal Co-IP, replicated across two papers (PMID:21558276 and PMID:21504906)","pmids":["21558276"],"is_preprint":false},{"year":2011,"finding":"RFWD3 directly interacts with RPA2 using purified proteins, is recruited to stalled replication forks, co-localizes with RPA2 under replication stress, and is required for ATR-dependent Chk1 activation. Deletion of the RPA2-binding region on RFWD3 impairs its localization to stalled forks and decreases Chk1 activation.","method":"Pulldown with purified proteins, immunofluorescence, deletion mutant analysis, Chk1 phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction with purified components plus functional epistasis via deletion mutant, single lab","pmids":["21504906"],"is_preprint":false},{"year":2015,"finding":"RFWD3 mediates multi-site ubiquitination of the entire RPA complex upon replication fork stalling. Ubiquitination occurs on chromatin at sites outside the DNA binding channel, does not cause proteasomal degradation, and increases under fork collapse conditions. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination at stalled replication forks.","method":"Quantitative proteomics (ubiquitin profiling), mutational analysis of RPA ubiquitination sites, HR assay, fork restart assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — quantitative proteomics plus mutagenesis plus functional HR/fork restart assays, replicated concept across multiple subsequent papers","pmids":["26474068"],"is_preprint":false},{"year":2017,"finding":"RFWD3 mutations cause Fanconi anemia (FANCW). An I639K substitution in the WD40 repeats abolishes interaction of RFWD3 with RPA, preventing RFWD3 recruitment to ICL-stalled replication forks. Single point mutations in RPA32 that abolish RFWD3 interaction also inhibit ICL repair. Unloading of RPA from ICL-induced sites is perturbed in RFWD3-deficient cells.","method":"Patient mutation analysis, Co-immunoprecipitation, site-directed mutagenesis, immunofluorescence, ICL repair assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal mutagenesis of both RFWD3 and RPA binding interface confirming the interaction, complemented by functional ICL repair assays, independently corroborated by companion paper PMID:28691929","pmids":["28575657"],"is_preprint":false},{"year":2017,"finding":"RFWD3 is the FA gene FANCW. Patient-derived mutations in RFWD3 impair its relocation to chromatin and physical interaction with RPA. RFWD3-mutant cells show defective HR, increased ICL sensitivity, G2 arrest, and chromosomal breakage. Rfwd3 knockout mice show increased embryonic lethality, subfertility, and gonadal atrophy.","method":"Patient cell complementation, engineered cell lines (human and avian), chromosomal breakage assay, HR assay, chromatin fractionation, Co-IP, mouse knockout model","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (complementation, HR assay, Co-IP, mouse model) across human cells and animal model, independently replicated in companion papers","pmids":["28691929"],"is_preprint":false},{"year":2017,"finding":"RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM is required for this ubiquitination activity in vivo. RFWD3 promotes VCP/p97-mediated protein dynamics and subsequent degradation of both RPA and RAD51, thereby removing them from DNA damage sites. MMC-induced chromatin loading of MCM8 and RAD54 is defective in RFWD3-inactivated cells or cells expressing ubiquitination-deficient RAD51, indicating RAD51 ubiquitination is needed for late-phase HR.","method":"In vitro ubiquitination assay, cell-based ubiquitination assay, immunofluorescence, chromatin fractionation, ubiquitination-deficient RAD51 mutant expression","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro ubiquitination reconstitution plus in vivo mutant rescue experiments with multiple orthogonal readouts in a single rigorous study","pmids":["28575658"],"is_preprint":false},{"year":2018,"finding":"RFWD3 localizes at replication forks in unperturbed cells and associates with PCNA via a PIP motif. PCNA association is critical for RFWD3 stability. Cells lacking RFWD3 show slower fork progression, prolonged S phase, and increased loading of replication fork components. The S-phase defect is rescued by WT RFWD3 but not by a PIP mutant. RFWD3-PCNA interaction enables polyubiquitination of RPA for proper DNA replication.","method":"Co-immunoprecipitation, PIP-motif mutant complementation, DNA fiber assay, chromatin fractionation, immunofluorescence","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — PIP-mutant rescue experiment plus DNA fiber analysis plus Co-IP, multiple orthogonal methods in a single study","pmids":["30530694"],"is_preprint":false},{"year":2020,"finding":"RFWD3 promotes ubiquitylation of proteins on ssDNA at stalled forks, which is required for PCNA ubiquitylation. Absence of RFWD3 leads to defective recruitment of key repair and signaling factors to damaged chromatin, inhibits PCNA ubiquitylation, and drastically impairs translesion DNA synthesis (TLS) across different DNA lesions.","method":"Mass spectrometry-based ubiquitin proteomics, chromatin fractionation, TLS assay, RFWD3 knockout/knockdown, PCNA ubiquitylation assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — mass spectrometry ubiquitin profiling plus functional TLS assays plus PCNA ubiquitylation assay, multiple orthogonal methods in a single study","pmids":["33321094"],"is_preprint":false},{"year":2020,"finding":"In BRCA2-deficient cells, stalled forks accumulate phosphorylated and ubiquitinated RPA (ubq-pRPA); the ubiquitination is mediated by RFWD3 and depends on SMARCAL1-dependent fork reversal. Co-depletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity in BRCA2-deficient cells.","method":"siRNA co-depletion, immunofluorescence, DNA fiber assay, Co-immunoprecipitation, ubiquitination assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistatic co-depletion with fork rescue readout plus ubiquitination assay, single lab with two orthogonal methods","pmids":["32391871"],"is_preprint":false},{"year":2020,"finding":"The E3 ligase activity of RFWD3 is required for stabilization of the Origin Recognition Complex (ORC) and ORC-Associated protein (ORCA/LRWD1) in cells expressing wild-type p53. RFWD3 associates with ORC/ORCA (Co-IP), and depletion of RFWD3 reduces ORC/ORCA protein levels. Overexpression of ORC/ORCA leads to stabilization of RFWD3.","method":"Co-immunoprecipitation, catalytic mutant analysis, siRNA knockdown, western blot","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and knockdown results from a single lab, catalytic mutant used but mechanism of ORC stabilization not biochemically reconstituted","pmids":["33044890"],"is_preprint":false},{"year":2020,"finding":"The Drosophila ortholog of RFWD3 (Mus302) functions independently of RAD51 during DNA repair, and evidence does not support a role for Mus302 in repair of collapsed replication forks or DSBs through HR — in contrast to the human protein. The N-terminal third of RFWD3 is present only in mammals, suggesting this domain underlies the acquisition of HR-related functions in mammals.","method":"Genetic mutant analysis, DNA repair assays in Drosophila","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function experiments in Drosophila showing negative result (no HR role) combined with sequence analysis identifying mammal-specific N-terminal domain","pmids":["31900333"],"is_preprint":false},{"year":2022,"finding":"RFWD3 contributes to PCNA modification-dependent DNA damage tolerance independently of both the Fanconi anemia pathway and DNA polymerase η. RFWD3's role in cellular survival after UV irradiation is dependent on PCNA modifications at K164.","method":"Genetic epistasis (RFWD3 knockout combined with PCNA K164 mutation), cell survival assay, TLS assay","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with clean KO cell lines and defined survival phenotype, single lab","pmids":["35905994"],"is_preprint":false},{"year":2023,"finding":"RFWD3 promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. RFWD3 stimulates fork remodeling (reversal) in a ZRANB3-epistatic manner. RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing the mechanism for ZRANB3 recruitment. Inactivation of RFWD3 in BRCA2-deficient cells rescues fork degradation analogously to ZRANB3 inactivation.","method":"Immunofluorescence, electron microscopy of replication forks, epistasis experiments (co-depletion), PCNA ubiquitination assay, Co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — electron microscopy fork reversal assay plus epistasis plus Co-IP/ubiquitination assay, multiple orthogonal methods establishing mechanistic pathway","pmids":["37036693"],"is_preprint":false},{"year":2025,"finding":"UHRF1 acts as an E3 ubiquitin ligase for RFWD3, ubiquitinating and destabilizing it to prevent premature RAD51 removal during HR. RAD51 in turn protects RFWD3 from UHRF1, establishing a negative feedback circuit. Phosphorylation of UHRF1 (regulated by phosphatase PP4) controls its E3 activity toward RFWD3.","method":"Co-immunoprecipitation, in vitro/in vivo ubiquitination assay, phosphorylation mutant analysis, PP4 knockdown","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ubiquitination assay and Co-IP from single lab; mechanistic circuit is compelling but not yet independently replicated","pmids":["40940676"],"is_preprint":false},{"year":2025,"finding":"RFWD3 competes with E3 ligase TRIM24 to stabilize TREX1 (a cytosolic dsDNA degrader) by sequestering it from TRIM24-mediated proteasomal degradation. RFWD3 inhibition increases intracellular dsDNA, activates STING-IFN signaling, decreases MDSCs, and enhances PD-L1 blockade efficacy. RFWD3 was identified as a TREX1-interacting protein by mass spectrometry.","method":"Mass spectrometry (TREX1 interactome), Co-immunoprecipitation, RFWD3 overexpression/inhibition in murine NSCLC models, STING pathway activation assay","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — mass spectrometry identification plus Co-IP and in vivo mouse model, single lab, novel finding not yet independently replicated","pmids":["41117130"],"is_preprint":false},{"year":2025,"finding":"SLFN11 antagonizes the RFWD3-PRIMPOL fork restart pathway by disrupting recruitment of both RFWD3 and PRIMPOL to stalled forks. In the absence of SLFN11, fork restart proceeds via RFWD3 and PRIMPOL to facilitate gapped DNA synthesis. SLFN11's suppression of RFWD3/PRIMPOL recruitment requires a functional ATPase domain and persistent fork localization, but not tRNA hydrolysis or ssDNA binding.","method":"Single-molecule DNA fiber analysis, super-resolution microscopy, RFWD3/PRIMPOL recruitment assay, SLFN11 ATPase mutant analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single-molecule fiber analysis plus super-resolution microscopy plus domain mutant epistasis, single lab with orthogonal methods","pmids":["41372167"],"is_preprint":false}],"current_model":"RFWD3 is a RING-finger/WD40-containing E3 ubiquitin ligase that is recruited to stalled replication forks via direct interaction with RPA (through its C-terminal WD40 domain) and stabilized there by PCNA through a PIP motif; it polyubiquitinates RPA and RAD51 (requiring ATR/ATM-mediated phosphorylation) to promote their VCP/p97-dependent removal, enabling progression through late-phase homologous recombination, replication fork restart, and translesion synthesis. It also drives PCNA ubiquitination at K164 to recruit ZRANB3 for fork reversal, forms a complex with Mdm2 to stabilize p53 after DNA damage, is itself regulated by UHRF1-mediated ubiquitination in a negative-feedback circuit with RAD51, and its loss causes Fanconi anemia complementation group W (FANCW)."},"narrative":{"mechanistic_narrative":"RFWD3 is a RING-finger/WD40 E3 ubiquitin ligase that governs the resolution and restart of stalled and damaged DNA replication forks by remodeling the protein composition of single-stranded DNA at sites of replication stress [PMID:26474068, PMID:33321094]. It is recruited to damaged chromatin and stalled forks through a direct interaction between its C-terminal coiled-coil/WD40 region and the RPA2 subunit of RPA, an interaction required for ATR-dependent Chk1 activation and for localization to interstrand crosslink (ICL)-stalled forks [PMID:21558276, PMID:21504906, PMID:28575657]. At ongoing forks RFWD3 also associates with PCNA via a PIP motif, an interaction that stabilizes the ligase and supports normal fork progression [PMID:30530694]. RFWD3 catalyzes multi-site polyubiquitination of the RPA complex and of RAD51, modifications that depend on ATR/ATM phosphorylation of RFWD3 and drive VCP/p97-mediated extraction of these factors from DNA, enabling late-phase homologous recombination, fork restart, and ICL repair [PMID:26474068, PMID:28575658]. By promoting PCNA ubiquitination, RFWD3 licenses translesion synthesis and recruits the DNA translocase ZRANB3 to catalyze fork reversal [PMID:33321094, PMID:37036693]. Biallelic RFWD3 mutations that disrupt RPA binding and chromatin recruitment cause Fanconi anemia complementation group W, with affected cells showing defective HR, ICL hypersensitivity, and chromosomal breakage [PMID:28575657, PMID:28691929]. Beyond the replication-stress core, RFWD3 cooperates with Mdm2 to stabilize p53 in the late DNA-damage response [PMID:20173098], and is itself controlled by UHRF1-mediated ubiquitination in a negative-feedback circuit with RAD51 [PMID:40940676].","teleology":[{"year":2010,"claim":"Established RFWD3 as a DNA-damage-responsive E3 ligase acting in the p53 pathway, the first functional context for the protein.","evidence":"Co-IP and in vitro ubiquitination with Mdm2/p53 plus ATM/ATR phosphorylation-mutant analysis","pmids":["20173098"],"confidence":"Medium","gaps":["In vitro assays from a single lab; physiological ubiquitin chain type on p53 not defined","Did not connect RFWD3 to replication-fork biology"]},{"year":2011,"claim":"Identified RPA (RPA2) as the direct recruitment platform that brings RFWD3 to damaged sites and stalled forks, explaining how the ligase is targeted to replication stress.","evidence":"Pulldown with purified proteins, reciprocal Co-IP, deletion-mutant immunofluorescence, and Chk1 phosphorylation assays across two papers","pmids":["21558276","21504906"],"confidence":"Medium","gaps":["Ubiquitination substrates at forks not yet defined","Mechanism linking recruitment to Chk1 activation unresolved"]},{"year":2015,"claim":"Showed RFWD3 multi-site ubiquitinates the RPA complex non-degradatively to drive fork restart and HR, defining its core enzymatic output at stalled forks.","evidence":"Quantitative ubiquitin proteomics, RPA site mutagenesis, HR and fork-restart assays","pmids":["26474068"],"confidence":"High","gaps":["Downstream reader/extraction machinery not yet identified in this study","Did not address RAD51"]},{"year":2017,"claim":"Linked RFWD3 to human disease as the Fanconi anemia gene FANCW and defined the RPA-binding interface as the critical disease determinant.","evidence":"Patient mutation analysis, reciprocal RFWD3/RPA32 mutagenesis, ICL repair assays, patient-cell complementation, chromosomal breakage assays, and Rfwd3 knockout mice","pmids":["28575657","28691929"],"confidence":"High","gaps":["Genotype-phenotype spectrum across patients not fully mapped","Embryonic lethality mechanism in mice not dissected"]},{"year":2017,"claim":"Demonstrated RFWD3 ubiquitinates both RPA and RAD51 to promote their p97/VCP-mediated removal, establishing the mechanism for late-phase HR completion.","evidence":"In vitro and cell-based ubiquitination assays, ubiquitination-deficient RAD51 mutant, chromatin fractionation of MCM8/RAD54","pmids":["28575658"],"confidence":"High","gaps":["Lysine specificity and chain architecture on RAD51 not fully resolved","Coordination between RPA and RAD51 ubiquitination timing unclear"]},{"year":2018,"claim":"Revealed a PCNA-PIP interaction that stabilizes RFWD3 and supports unperturbed replication, extending its role beyond damage to normal S phase.","evidence":"Co-IP, PIP-mutant complementation, DNA fiber assay, chromatin fractionation","pmids":["30530694"],"confidence":"High","gaps":["How PCNA binding is coordinated with RPA recruitment not defined","Identity of all PIP-dependent substrates incomplete"]},{"year":2020,"claim":"Connected RFWD3-driven ssDNA-protein ubiquitination to PCNA ubiquitylation and translesion synthesis, broadening its role to DNA damage tolerance.","evidence":"Ubiquitin proteomics, chromatin fractionation, TLS assays, PCNA ubiquitylation assays in knockout/knockdown cells","pmids":["33321094"],"confidence":"High","gaps":["Direct vs indirect role in PCNA ubiquitination not fully separated","Which ssDNA substrates are functionally critical for TLS unresolved"]},{"year":2020,"claim":"Placed RFWD3 in fork-protection biology by showing its RPA ubiquitination drives fork degradation in BRCA2-deficient cells via SMARCAL1-dependent reversal.","evidence":"siRNA co-depletion, DNA fiber, Co-IP, ubiquitination assays","pmids":["32391871"],"confidence":"Medium","gaps":["Single lab; nuclease responsible for degradation downstream not defined here","Relationship to RAD51 removal at protected forks unclear"]},{"year":2020,"claim":"Proposed an additional RFWD3 role in stabilizing the origin recognition complex in a p53-dependent manner.","evidence":"Co-IP, catalytic-mutant analysis, siRNA knockdown, western blot","pmids":["33044890"],"confidence":"Low","gaps":["Single Co-IP/knockdown lab study; ORC stabilization mechanism not biochemically reconstituted","Whether ORC is a direct ubiquitination substrate unknown"]},{"year":2020,"claim":"Used the Drosophila ortholog to argue the mammalian HR-related functions map to a mammal-specific N-terminal domain absent in flies.","evidence":"Genetic mutant analysis and DNA repair assays in Drosophila plus sequence comparison","pmids":["31900333"],"confidence":"Medium","gaps":["Direct functional mapping of the N-terminal domain in human protein not performed","Negative result in fly does not exclude conserved partial functions"]},{"year":2022,"claim":"Defined RFWD3's damage-tolerance role as dependent on PCNA K164 modification but independent of the canonical FA pathway and pol eta.","evidence":"Genetic epistasis of RFWD3 knockout with PCNA K164 mutation, survival and TLS assays","pmids":["35905994"],"confidence":"Medium","gaps":["Which TLS polymerases substitute for pol eta downstream unknown","Single lab"]},{"year":2023,"claim":"Mechanistically linked RFWD3-driven PCNA ubiquitination to ZRANB3 recruitment and fork reversal, unifying its ubiquitination output with fork remodeling.","evidence":"Electron microscopy of forks, co-depletion epistasis, PCNA ubiquitination assays, Co-IP","pmids":["37036693"],"confidence":"High","gaps":["Whether RPA/RAD51 ubiquitination and ZRANB3-PCNA ubiquitination are temporally coupled unclear","Other translocases acting in parallel not excluded"]},{"year":2025,"claim":"Identified UHRF1-mediated ubiquitination of RFWD3 as a negative-feedback brake protecting RAD51 from premature removal during HR.","evidence":"Co-IP, in vitro/in vivo ubiquitination, phosphorylation-mutant and PP4 knockdown analysis","pmids":["40940676"],"confidence":"Medium","gaps":["Single lab, not independently replicated","Physiological trigger timing of the feedback circuit undefined"]},{"year":2025,"claim":"Revealed a non-replication role in which RFWD3 stabilizes TREX1 by competing with TRIM24, linking it to cytosolic dsDNA sensing and STING-IFN immune signaling.","evidence":"TREX1 interactome mass spectrometry, Co-IP, RFWD3 overexpression/inhibition in murine NSCLC models, STING activation assays","pmids":["41117130"],"confidence":"Medium","gaps":["Single lab, not independently replicated","Whether RFWD3 ubiquitinates TREX1 directly or acts only by sequestration unclear"]},{"year":2025,"claim":"Positioned RFWD3 within a fork-restart pathway antagonized by SLFN11, defining an upstream regulatory layer for its recruitment.","evidence":"Single-molecule DNA fiber analysis, super-resolution microscopy, SLFN11 ATPase-domain mutant epistasis","pmids":["41372167"],"confidence":"Medium","gaps":["Mechanism by which SLFN11 blocks RFWD3/PRIMPOL recruitment not biochemically defined","Single lab"]},{"year":null,"claim":"How RFWD3's multiple ubiquitination outputs (RPA, RAD51, PCNA, p53, TREX1) are temporally and spatially coordinated, and what determines substrate choice at a given fork, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of RFWD3 substrate engagement","Substrate-selection logic governing competing ubiquitination events undefined","Direct versus indirect substrates incompletely separated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,3,6,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,6,8,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,5]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[5,7,8]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[3,4,6]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[7,8,13]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,6]}],"complexes":[],"partners":["RPA2","RAD51","PCNA","MDM2","ZRANB3","UHRF1","TREX1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6PCD5","full_name":"E3 ubiquitin-protein ligase RFWD3","aliases":["RING finger and WD repeat domain-containing protein 3","RING finger protein 201"],"length_aa":774,"mass_kda":85.1,"function":"E3 ubiquitin-protein ligase required for the repair of DNA interstrand cross-links (ICL) in response to DNA damage (PubMed:21504906, PubMed:21558276, PubMed:26474068, PubMed:28575657, PubMed:28575658, PubMed:33321094). Plays a key role in RPA-mediated DNA damage signaling and repair (PubMed:21504906, PubMed:21558276, PubMed:26474068, PubMed:28575657, PubMed:28575658, PubMed:28691929). Acts by mediating ubiquitination of the RPA complex (RPA1, RPA2 and RPA3 subunits) and RAD51 at stalled replication forks, leading to remove them from DNA damage sites and promote homologous recombination (PubMed:26474068, PubMed:28575657, PubMed:28575658). Also mediates the ubiquitination of p53/TP53 in the late response to DNA damage, and acts as a positive regulator of p53/TP53 stability, thereby regulating the G1/S DNA damage checkpoint (PubMed:20173098). May act by catalyzing the formation of short polyubiquitin chains on p53/TP53 that are not targeted to the proteasome (PubMed:20173098). In response to ionizing radiation, interacts with MDM2 and enhances p53/TP53 ubiquitination, possibly by restricting MDM2 from extending polyubiquitin chains on ubiquitinated p53/TP53 (PubMed:20173098). Required to translesion DNA synthesis across DNA-protein cross-link adducts by catalyzing ubiquitination of proteins on single-stranded DNA (ssDNA) (PubMed:33321094)","subcellular_location":"Nucleus; Nucleus, PML body; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q6PCD5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RFWD3","classification":"Not Classified","n_dependent_lines":201,"n_total_lines":1208,"dependency_fraction":0.16639072847682118},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CSNK2B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RFWD3","total_profiled":1310},"omim":[{"mim_id":"617784","title":"FANCONI ANEMIA, COMPLEMENTATION GROUP W; FANCW","url":"https://www.omim.org/entry/617784"},{"mim_id":"614151","title":"RING FINGER AND WD REPEAT DOMAINS-CONTAINING PROTEIN 3; RFWD3","url":"https://www.omim.org/entry/614151"},{"mim_id":"227650","title":"FANCONI ANEMIA, COMPLEMENTATION GROUP A; FANCA","url":"https://www.omim.org/entry/227650"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":44.9}],"url":"https://www.proteinatlas.org/search/RFWD3"},"hgnc":{"alias_symbol":["FLJ10520","RNF201","FANCW"],"prev_symbol":[]},"alphafold":{"accession":"Q6PCD5","domains":[{"cath_id":"3.30.40.10","chopping":"286-344","consensus_level":"medium","plddt":90.3527,"start":286,"end":344},{"cath_id":"2.130.10.10","chopping":"437-774","consensus_level":"medium","plddt":92.9154,"start":437,"end":774}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PCD5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PCD5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6PCD5-F1-predicted_aligned_error_v6.png","plddt_mean":70.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RFWD3","jax_strain_url":"https://www.jax.org/strain/search?query=RFWD3"},"sequence":{"accession":"Q6PCD5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6PCD5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6PCD5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6PCD5"}},"corpus_meta":[{"pmid":"28691929","id":"PMC_28691929","title":"Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia.","date":"2017","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/28691929","citation_count":163,"is_preprint":false},{"pmid":"28575658","id":"PMC_28575658","title":"RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.","date":"2017","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/28575658","citation_count":142,"is_preprint":false},{"pmid":"26474068","id":"PMC_26474068","title":"RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.","date":"2015","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/26474068","citation_count":115,"is_preprint":false},{"pmid":"33321094","id":"PMC_33321094","title":"The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.","date":"2020","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/33321094","citation_count":70,"is_preprint":false},{"pmid":"20173098","id":"PMC_20173098","title":"RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage.","date":"2010","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/20173098","citation_count":70,"is_preprint":false},{"pmid":"21558276","id":"PMC_21558276","title":"RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21558276","citation_count":66,"is_preprint":false},{"pmid":"28575657","id":"PMC_28575657","title":"RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.","date":"2017","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/28575657","citation_count":63,"is_preprint":false},{"pmid":"21504906","id":"PMC_21504906","title":"E3 ligase RFWD3 participates in replication checkpoint control.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21504906","citation_count":48,"is_preprint":false},{"pmid":"30530694","id":"PMC_30530694","title":"PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication.","date":"2018","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/30530694","citation_count":31,"is_preprint":false},{"pmid":"37036693","id":"PMC_37036693","title":"RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.","date":"2023","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/37036693","citation_count":21,"is_preprint":false},{"pmid":"32391871","id":"PMC_32391871","title":"E3 ligase RFWD3 is a novel modulator of stalled fork stability in BRCA2-deficient cells.","date":"2020","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/32391871","citation_count":21,"is_preprint":false},{"pmid":"26568529","id":"PMC_26568529","title":"Variant in the RFWD3 gene associated with PATN1, a modifier of leopard complex spotting.","date":"2015","source":"Animal genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26568529","citation_count":17,"is_preprint":false},{"pmid":"31571161","id":"PMC_31571161","title":"Identification of an E3 ligase-encoding gene RFWD3 in non-small cell lung cancer.","date":"2019","source":"Frontiers of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31571161","citation_count":14,"is_preprint":false},{"pmid":"34712656","id":"PMC_34712656","title":"RFWD3 Participates in the Occurrence and Development of Colorectal Cancer via E2F1 Transcriptional Regulation of BIRC5.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/34712656","citation_count":13,"is_preprint":false},{"pmid":"33842359","id":"PMC_33842359","title":"The Valproate Mediates Radio-Bidirectional Regulation Through RFWD3-Dependent Ubiquitination on Rad51.","date":"2021","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33842359","citation_count":13,"is_preprint":false},{"pmid":"32902405","id":"PMC_32902405","title":"Down-regulation of RFWD3 inhibits cancer cells proliferation and migration in gastric carcinoma.","date":"2020","source":"General physiology and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/32902405","citation_count":11,"is_preprint":false},{"pmid":"35905994","id":"PMC_35905994","title":"RFWD3 and translesion DNA polymerases contribute to PCNA modification-dependent DNA damage tolerance.","date":"2022","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/35905994","citation_count":8,"is_preprint":false},{"pmid":"33044890","id":"PMC_33044890","title":"The E3 ligase RFWD3 stabilizes ORC in a p53-dependent manner.","date":"2020","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/33044890","citation_count":8,"is_preprint":false},{"pmid":"33241105","id":"PMC_33241105","title":"RPA, RFWD3 and BRCA2 at stalled forks: a balancing act.","date":"2020","source":"Molecular & cellular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33241105","citation_count":6,"is_preprint":false},{"pmid":"36458571","id":"PMC_36458571","title":"RFWD3 acts as a tumor promotor in the development and progression of bladder cancer.","date":"2022","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/36458571","citation_count":5,"is_preprint":false},{"pmid":"38504674","id":"PMC_38504674","title":"MicroRNA-190b Targets RFWD3 in Estrogen Receptor-Positive Breast Cancer.","date":"2024","source":"Breast cancer : basic and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/38504674","citation_count":3,"is_preprint":false},{"pmid":"38711848","id":"PMC_38711848","title":"RFWD3 modulates response to platinum chemotherapy and promotes cancer associated phenotypes in high grade serous ovarian cancer.","date":"2024","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38711848","citation_count":3,"is_preprint":false},{"pmid":"41117130","id":"PMC_41117130","title":"Smohaze-Upregulated RFWD3 Competes with TRIM24 to Stabilize TREX1 and Reduce Cytosolic dsDNA in Non-Small Cell Lung Cancer.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41117130","citation_count":2,"is_preprint":false},{"pmid":"40940676","id":"PMC_40940676","title":"UHRF1 Controls the Timing of RAD51 Removal During DNA Damage Repair Through Suppressing RFWD3.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/40940676","citation_count":2,"is_preprint":false},{"pmid":"31900333","id":"PMC_31900333","title":"The Drosophila melanogaster Ortholog of RFWD3 Functions Independently of RAD51 During DNA Repair.","date":"2020","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/31900333","citation_count":2,"is_preprint":false},{"pmid":"41372167","id":"PMC_41372167","title":"SLFN11 counteracts the RFWD3-PRIMPOL DNA damage tolerance axis to restrain gapped DNA synthesis in response to replication stress.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41372167","citation_count":1,"is_preprint":false},{"pmid":"38058754","id":"PMC_38058754","title":"Further Evidence for RFWD3 Gene Causing Fanconi Anemia Complementation Group W: Detailed Clinical Report of the Second Case in the Literature.","date":"2023","source":"Molecular syndromology","url":"https://pubmed.ncbi.nlm.nih.gov/38058754","citation_count":1,"is_preprint":false},{"pmid":"40523204","id":"PMC_40523204","title":"RFWD3 Knockdown Inhibits Cancer Cell Proliferation, Migration, and Invasion while Promoting Apoptosis of Non-Small Cell Lung Cancer Cells.","date":"2025","source":"Archivum immunologiae et therapiae experimentalis","url":"https://pubmed.ncbi.nlm.nih.gov/40523204","citation_count":0,"is_preprint":false},{"pmid":"40700452","id":"PMC_40700452","title":"Knocking down RFWD3 suppresses the growth and migration of glioblastoma cells by blocking G2/M cycle progression.","date":"2026","source":"Journal of neuropathology and experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/40700452","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.24.666583","title":"The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses","date":"2025-07-26","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.24.666583","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15138,"output_tokens":4667,"usd":0.057709,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12788,"output_tokens":4559,"usd":0.088957,"stage2_stop_reason":"end_turn"},"total_usd":0.146666,"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\": 2010,\n      \"finding\": \"RFWD3 forms a complex with Mdm2 and p53, synergistically ubiquitinates p53 in vitro, and is required to stabilize p53 in the late response to DNA damage. RFWD3-Mdm2 complex restricts polyubiquitination of p53 by Mdm2 alone. RFWD3 is phosphorylated by ATM/ATR kinases, and phosphorylation mutants fail to stimulate p53 ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, phosphorylation-deficient mutant analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Weak — in vitro ubiquitination assay and Co-IP from a single lab with two orthogonal methods\",\n      \"pmids\": [\"20173098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RFWD3 directly interacts with RPA (specifically RPA2 subunit) and is recruited to sites of DNA damage in a RPA-dependent manner. The C-terminus of RFWD3 (coiled-coil and WD40 domains) is necessary for RPA binding. Loss of RFWD3 results in persistent γH2AX and RAD51 foci in damaged cells.\",\n      \"method\": \"Co-immunoprecipitation, pulldown with purified proteins, immunofluorescence, deletion mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction confirmed with purified proteins and reciprocal Co-IP, replicated across two papers (PMID:21558276 and PMID:21504906)\",\n      \"pmids\": [\"21558276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RFWD3 directly interacts with RPA2 using purified proteins, is recruited to stalled replication forks, co-localizes with RPA2 under replication stress, and is required for ATR-dependent Chk1 activation. Deletion of the RPA2-binding region on RFWD3 impairs its localization to stalled forks and decreases Chk1 activation.\",\n      \"method\": \"Pulldown with purified proteins, immunofluorescence, deletion mutant analysis, Chk1 phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction with purified components plus functional epistasis via deletion mutant, single lab\",\n      \"pmids\": [\"21504906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RFWD3 mediates multi-site ubiquitination of the entire RPA complex upon replication fork stalling. Ubiquitination occurs on chromatin at sites outside the DNA binding channel, does not cause proteasomal degradation, and increases under fork collapse conditions. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination at stalled replication forks.\",\n      \"method\": \"Quantitative proteomics (ubiquitin profiling), mutational analysis of RPA ubiquitination sites, HR assay, fork restart assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — quantitative proteomics plus mutagenesis plus functional HR/fork restart assays, replicated concept across multiple subsequent papers\",\n      \"pmids\": [\"26474068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RFWD3 mutations cause Fanconi anemia (FANCW). An I639K substitution in the WD40 repeats abolishes interaction of RFWD3 with RPA, preventing RFWD3 recruitment to ICL-stalled replication forks. Single point mutations in RPA32 that abolish RFWD3 interaction also inhibit ICL repair. Unloading of RPA from ICL-induced sites is perturbed in RFWD3-deficient cells.\",\n      \"method\": \"Patient mutation analysis, Co-immunoprecipitation, site-directed mutagenesis, immunofluorescence, ICL repair assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal mutagenesis of both RFWD3 and RPA binding interface confirming the interaction, complemented by functional ICL repair assays, independently corroborated by companion paper PMID:28691929\",\n      \"pmids\": [\"28575657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RFWD3 is the FA gene FANCW. Patient-derived mutations in RFWD3 impair its relocation to chromatin and physical interaction with RPA. RFWD3-mutant cells show defective HR, increased ICL sensitivity, G2 arrest, and chromosomal breakage. Rfwd3 knockout mice show increased embryonic lethality, subfertility, and gonadal atrophy.\",\n      \"method\": \"Patient cell complementation, engineered cell lines (human and avian), chromosomal breakage assay, HR assay, chromatin fractionation, Co-IP, mouse knockout model\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (complementation, HR assay, Co-IP, mouse model) across human cells and animal model, independently replicated in companion papers\",\n      \"pmids\": [\"28691929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM is required for this ubiquitination activity in vivo. RFWD3 promotes VCP/p97-mediated protein dynamics and subsequent degradation of both RPA and RAD51, thereby removing them from DNA damage sites. MMC-induced chromatin loading of MCM8 and RAD54 is defective in RFWD3-inactivated cells or cells expressing ubiquitination-deficient RAD51, indicating RAD51 ubiquitination is needed for late-phase HR.\",\n      \"method\": \"In vitro ubiquitination assay, cell-based ubiquitination assay, immunofluorescence, chromatin fractionation, ubiquitination-deficient RAD51 mutant expression\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro ubiquitination reconstitution plus in vivo mutant rescue experiments with multiple orthogonal readouts in a single rigorous study\",\n      \"pmids\": [\"28575658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RFWD3 localizes at replication forks in unperturbed cells and associates with PCNA via a PIP motif. PCNA association is critical for RFWD3 stability. Cells lacking RFWD3 show slower fork progression, prolonged S phase, and increased loading of replication fork components. The S-phase defect is rescued by WT RFWD3 but not by a PIP mutant. RFWD3-PCNA interaction enables polyubiquitination of RPA for proper DNA replication.\",\n      \"method\": \"Co-immunoprecipitation, PIP-motif mutant complementation, DNA fiber assay, chromatin fractionation, immunofluorescence\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PIP-mutant rescue experiment plus DNA fiber analysis plus Co-IP, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"30530694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RFWD3 promotes ubiquitylation of proteins on ssDNA at stalled forks, which is required for PCNA ubiquitylation. Absence of RFWD3 leads to defective recruitment of key repair and signaling factors to damaged chromatin, inhibits PCNA ubiquitylation, and drastically impairs translesion DNA synthesis (TLS) across different DNA lesions.\",\n      \"method\": \"Mass spectrometry-based ubiquitin proteomics, chromatin fractionation, TLS assay, RFWD3 knockout/knockdown, PCNA ubiquitylation assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mass spectrometry ubiquitin profiling plus functional TLS assays plus PCNA ubiquitylation assay, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"33321094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In BRCA2-deficient cells, stalled forks accumulate phosphorylated and ubiquitinated RPA (ubq-pRPA); the ubiquitination is mediated by RFWD3 and depends on SMARCAL1-dependent fork reversal. Co-depletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity in BRCA2-deficient cells.\",\n      \"method\": \"siRNA co-depletion, immunofluorescence, DNA fiber assay, Co-immunoprecipitation, ubiquitination assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistatic co-depletion with fork rescue readout plus ubiquitination assay, single lab with two orthogonal methods\",\n      \"pmids\": [\"32391871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The E3 ligase activity of RFWD3 is required for stabilization of the Origin Recognition Complex (ORC) and ORC-Associated protein (ORCA/LRWD1) in cells expressing wild-type p53. RFWD3 associates with ORC/ORCA (Co-IP), and depletion of RFWD3 reduces ORC/ORCA protein levels. Overexpression of ORC/ORCA leads to stabilization of RFWD3.\",\n      \"method\": \"Co-immunoprecipitation, catalytic mutant analysis, siRNA knockdown, western blot\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and knockdown results from a single lab, catalytic mutant used but mechanism of ORC stabilization not biochemically reconstituted\",\n      \"pmids\": [\"33044890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The Drosophila ortholog of RFWD3 (Mus302) functions independently of RAD51 during DNA repair, and evidence does not support a role for Mus302 in repair of collapsed replication forks or DSBs through HR — in contrast to the human protein. The N-terminal third of RFWD3 is present only in mammals, suggesting this domain underlies the acquisition of HR-related functions in mammals.\",\n      \"method\": \"Genetic mutant analysis, DNA repair assays in Drosophila\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function experiments in Drosophila showing negative result (no HR role) combined with sequence analysis identifying mammal-specific N-terminal domain\",\n      \"pmids\": [\"31900333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RFWD3 contributes to PCNA modification-dependent DNA damage tolerance independently of both the Fanconi anemia pathway and DNA polymerase η. RFWD3's role in cellular survival after UV irradiation is dependent on PCNA modifications at K164.\",\n      \"method\": \"Genetic epistasis (RFWD3 knockout combined with PCNA K164 mutation), cell survival assay, TLS assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with clean KO cell lines and defined survival phenotype, single lab\",\n      \"pmids\": [\"35905994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RFWD3 promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. RFWD3 stimulates fork remodeling (reversal) in a ZRANB3-epistatic manner. RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing the mechanism for ZRANB3 recruitment. Inactivation of RFWD3 in BRCA2-deficient cells rescues fork degradation analogously to ZRANB3 inactivation.\",\n      \"method\": \"Immunofluorescence, electron microscopy of replication forks, epistasis experiments (co-depletion), PCNA ubiquitination assay, Co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — electron microscopy fork reversal assay plus epistasis plus Co-IP/ubiquitination assay, multiple orthogonal methods establishing mechanistic pathway\",\n      \"pmids\": [\"37036693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UHRF1 acts as an E3 ubiquitin ligase for RFWD3, ubiquitinating and destabilizing it to prevent premature RAD51 removal during HR. RAD51 in turn protects RFWD3 from UHRF1, establishing a negative feedback circuit. Phosphorylation of UHRF1 (regulated by phosphatase PP4) controls its E3 activity toward RFWD3.\",\n      \"method\": \"Co-immunoprecipitation, in vitro/in vivo ubiquitination assay, phosphorylation mutant analysis, PP4 knockdown\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ubiquitination assay and Co-IP from single lab; mechanistic circuit is compelling but not yet independently replicated\",\n      \"pmids\": [\"40940676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFWD3 competes with E3 ligase TRIM24 to stabilize TREX1 (a cytosolic dsDNA degrader) by sequestering it from TRIM24-mediated proteasomal degradation. RFWD3 inhibition increases intracellular dsDNA, activates STING-IFN signaling, decreases MDSCs, and enhances PD-L1 blockade efficacy. RFWD3 was identified as a TREX1-interacting protein by mass spectrometry.\",\n      \"method\": \"Mass spectrometry (TREX1 interactome), Co-immunoprecipitation, RFWD3 overexpression/inhibition in murine NSCLC models, STING pathway activation assay\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — mass spectrometry identification plus Co-IP and in vivo mouse model, single lab, novel finding not yet independently replicated\",\n      \"pmids\": [\"41117130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SLFN11 antagonizes the RFWD3-PRIMPOL fork restart pathway by disrupting recruitment of both RFWD3 and PRIMPOL to stalled forks. In the absence of SLFN11, fork restart proceeds via RFWD3 and PRIMPOL to facilitate gapped DNA synthesis. SLFN11's suppression of RFWD3/PRIMPOL recruitment requires a functional ATPase domain and persistent fork localization, but not tRNA hydrolysis or ssDNA binding.\",\n      \"method\": \"Single-molecule DNA fiber analysis, super-resolution microscopy, RFWD3/PRIMPOL recruitment assay, SLFN11 ATPase mutant analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single-molecule fiber analysis plus super-resolution microscopy plus domain mutant epistasis, single lab with orthogonal methods\",\n      \"pmids\": [\"41372167\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RFWD3 is a RING-finger/WD40-containing E3 ubiquitin ligase that is recruited to stalled replication forks via direct interaction with RPA (through its C-terminal WD40 domain) and stabilized there by PCNA through a PIP motif; it polyubiquitinates RPA and RAD51 (requiring ATR/ATM-mediated phosphorylation) to promote their VCP/p97-dependent removal, enabling progression through late-phase homologous recombination, replication fork restart, and translesion synthesis. It also drives PCNA ubiquitination at K164 to recruit ZRANB3 for fork reversal, forms a complex with Mdm2 to stabilize p53 after DNA damage, is itself regulated by UHRF1-mediated ubiquitination in a negative-feedback circuit with RAD51, and its loss causes Fanconi anemia complementation group W (FANCW).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RFWD3 is a RING-finger/WD40 E3 ubiquitin ligase that governs the resolution and restart of stalled and damaged DNA replication forks by remodeling the protein composition of single-stranded DNA at sites of replication stress [#3, #8]. It is recruited to damaged chromatin and stalled forks through a direct interaction between its C-terminal coiled-coil/WD40 region and the RPA2 subunit of RPA, an interaction required for ATR-dependent Chk1 activation and for localization to interstrand crosslink (ICL)-stalled forks [#1, #2, #4]. At ongoing forks RFWD3 also associates with PCNA via a PIP motif, an interaction that stabilizes the ligase and supports normal fork progression [#7]. RFWD3 catalyzes multi-site polyubiquitination of the RPA complex and of RAD51, modifications that depend on ATR/ATM phosphorylation of RFWD3 and drive VCP/p97-mediated extraction of these factors from DNA, enabling late-phase homologous recombination, fork restart, and ICL repair [#3, #6]. By promoting PCNA ubiquitination, RFWD3 licenses translesion synthesis and recruits the DNA translocase ZRANB3 to catalyze fork reversal [#8, #13]. Biallelic RFWD3 mutations that disrupt RPA binding and chromatin recruitment cause Fanconi anemia complementation group W, with affected cells showing defective HR, ICL hypersensitivity, and chromosomal breakage [#4, #5]. Beyond the replication-stress core, RFWD3 cooperates with Mdm2 to stabilize p53 in the late DNA-damage response [#0], and is itself controlled by UHRF1-mediated ubiquitination in a negative-feedback circuit with RAD51 [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established RFWD3 as a DNA-damage-responsive E3 ligase acting in the p53 pathway, the first functional context for the protein.\",\n      \"evidence\": \"Co-IP and in vitro ubiquitination with Mdm2/p53 plus ATM/ATR phosphorylation-mutant analysis\",\n      \"pmids\": [\"20173098\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro assays from a single lab; physiological ubiquitin chain type on p53 not defined\", \"Did not connect RFWD3 to replication-fork biology\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified RPA (RPA2) as the direct recruitment platform that brings RFWD3 to damaged sites and stalled forks, explaining how the ligase is targeted to replication stress.\",\n      \"evidence\": \"Pulldown with purified proteins, reciprocal Co-IP, deletion-mutant immunofluorescence, and Chk1 phosphorylation assays across two papers\",\n      \"pmids\": [\"21558276\", \"21504906\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination substrates at forks not yet defined\", \"Mechanism linking recruitment to Chk1 activation unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed RFWD3 multi-site ubiquitinates the RPA complex non-degradatively to drive fork restart and HR, defining its core enzymatic output at stalled forks.\",\n      \"evidence\": \"Quantitative ubiquitin proteomics, RPA site mutagenesis, HR and fork-restart assays\",\n      \"pmids\": [\"26474068\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream reader/extraction machinery not yet identified in this study\", \"Did not address RAD51\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked RFWD3 to human disease as the Fanconi anemia gene FANCW and defined the RPA-binding interface as the critical disease determinant.\",\n      \"evidence\": \"Patient mutation analysis, reciprocal RFWD3/RPA32 mutagenesis, ICL repair assays, patient-cell complementation, chromosomal breakage assays, and Rfwd3 knockout mice\",\n      \"pmids\": [\"28575657\", \"28691929\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genotype-phenotype spectrum across patients not fully mapped\", \"Embryonic lethality mechanism in mice not dissected\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated RFWD3 ubiquitinates both RPA and RAD51 to promote their p97/VCP-mediated removal, establishing the mechanism for late-phase HR completion.\",\n      \"evidence\": \"In vitro and cell-based ubiquitination assays, ubiquitination-deficient RAD51 mutant, chromatin fractionation of MCM8/RAD54\",\n      \"pmids\": [\"28575658\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lysine specificity and chain architecture on RAD51 not fully resolved\", \"Coordination between RPA and RAD51 ubiquitination timing unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed a PCNA-PIP interaction that stabilizes RFWD3 and supports unperturbed replication, extending its role beyond damage to normal S phase.\",\n      \"evidence\": \"Co-IP, PIP-mutant complementation, DNA fiber assay, chromatin fractionation\",\n      \"pmids\": [\"30530694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PCNA binding is coordinated with RPA recruitment not defined\", \"Identity of all PIP-dependent substrates incomplete\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected RFWD3-driven ssDNA-protein ubiquitination to PCNA ubiquitylation and translesion synthesis, broadening its role to DNA damage tolerance.\",\n      \"evidence\": \"Ubiquitin proteomics, chromatin fractionation, TLS assays, PCNA ubiquitylation assays in knockout/knockdown cells\",\n      \"pmids\": [\"33321094\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect role in PCNA ubiquitination not fully separated\", \"Which ssDNA substrates are functionally critical for TLS unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed RFWD3 in fork-protection biology by showing its RPA ubiquitination drives fork degradation in BRCA2-deficient cells via SMARCAL1-dependent reversal.\",\n      \"evidence\": \"siRNA co-depletion, DNA fiber, Co-IP, ubiquitination assays\",\n      \"pmids\": [\"32391871\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; nuclease responsible for degradation downstream not defined here\", \"Relationship to RAD51 removal at protected forks unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Proposed an additional RFWD3 role in stabilizing the origin recognition complex in a p53-dependent manner.\",\n      \"evidence\": \"Co-IP, catalytic-mutant analysis, siRNA knockdown, western blot\",\n      \"pmids\": [\"33044890\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP/knockdown lab study; ORC stabilization mechanism not biochemically reconstituted\", \"Whether ORC is a direct ubiquitination substrate unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Used the Drosophila ortholog to argue the mammalian HR-related functions map to a mammal-specific N-terminal domain absent in flies.\",\n      \"evidence\": \"Genetic mutant analysis and DNA repair assays in Drosophila plus sequence comparison\",\n      \"pmids\": [\"31900333\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct functional mapping of the N-terminal domain in human protein not performed\", \"Negative result in fly does not exclude conserved partial functions\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined RFWD3's damage-tolerance role as dependent on PCNA K164 modification but independent of the canonical FA pathway and pol eta.\",\n      \"evidence\": \"Genetic epistasis of RFWD3 knockout with PCNA K164 mutation, survival and TLS assays\",\n      \"pmids\": [\"35905994\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which TLS polymerases substitute for pol eta downstream unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mechanistically linked RFWD3-driven PCNA ubiquitination to ZRANB3 recruitment and fork reversal, unifying its ubiquitination output with fork remodeling.\",\n      \"evidence\": \"Electron microscopy of forks, co-depletion epistasis, PCNA ubiquitination assays, Co-IP\",\n      \"pmids\": [\"37036693\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RPA/RAD51 ubiquitination and ZRANB3-PCNA ubiquitination are temporally coupled unclear\", \"Other translocases acting in parallel not excluded\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified UHRF1-mediated ubiquitination of RFWD3 as a negative-feedback brake protecting RAD51 from premature removal during HR.\",\n      \"evidence\": \"Co-IP, in vitro/in vivo ubiquitination, phosphorylation-mutant and PP4 knockdown analysis\",\n      \"pmids\": [\"40940676\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, not independently replicated\", \"Physiological trigger timing of the feedback circuit undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a non-replication role in which RFWD3 stabilizes TREX1 by competing with TRIM24, linking it to cytosolic dsDNA sensing and STING-IFN immune signaling.\",\n      \"evidence\": \"TREX1 interactome mass spectrometry, Co-IP, RFWD3 overexpression/inhibition in murine NSCLC models, STING activation assays\",\n      \"pmids\": [\"41117130\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, not independently replicated\", \"Whether RFWD3 ubiquitinates TREX1 directly or acts only by sequestration unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Positioned RFWD3 within a fork-restart pathway antagonized by SLFN11, defining an upstream regulatory layer for its recruitment.\",\n      \"evidence\": \"Single-molecule DNA fiber analysis, super-resolution microscopy, SLFN11 ATPase-domain mutant epistasis\",\n      \"pmids\": [\"41372167\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which SLFN11 blocks RFWD3/PRIMPOL recruitment not biochemically defined\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RFWD3's multiple ubiquitination outputs (RPA, RAD51, PCNA, p53, TREX1) are temporally and spatially coordinated, and what determines substrate choice at a given fork, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of RFWD3 substrate engagement\", \"Substrate-selection logic governing competing ubiquitination events undefined\", \"Direct versus indirect substrates incompletely separated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0061630\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 3, 6, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 6, 8, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [5, 7, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [3, 4, 6]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [7, 8, 13]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RPA2\", \"RAD51\", \"PCNA\", \"MDM2\", \"ZRANB3\", \"UHRF1\", \"TREX1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}