{"gene":"TOP3B","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2014,"finding":"TOP3B forms a complex with TDRD3, which acts as a molecular bridge between TOP3B and arginine-methylated histones (H4R3me2a). The TDRD3-TOP3B complex is recruited to the c-MYC gene promoter, where TOP3B relaxes negatively supercoiled DNA and reduces transcription-generated R-loops in vitro. TDRD3 knockdown in cells increases R-loop formation at the c-MYC locus, and Tdrd3 null mice show elevated R-loop formation and increased c-Myc/Igh chromosomal translocations.","method":"Co-immunoprecipitation, in vitro topoisomerase assay, R-loop detection (DRIP), ChIP, Tdrd3 knockout mouse model","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro biochemical assays, genetic knockout mouse model with multiple orthogonal readouts across labs","pmids":["24507716"],"is_preprint":false},{"year":2018,"finding":"TOP3B is arginine-methylated at R833 and R835 in its C-terminal RGG motif by PRMT1, PRMT3, and PRMT6 in vitro. Methylation-deficient TOP3B (R833/835K) shows reduced relaxation of negatively supercoiled DNA and increased R-loop accumulation in vitro and in cells. Additionally, methylation at R833/835 is partially required for TOP3B interaction with TDRD3 and for TOP3B localization to stress granules.","method":"In vitro methylation assay, site-directed mutagenesis, methylation-specific antibody, in vitro topoisomerase assay, R-loop detection, immunofluorescence for stress granule localization, Co-IP","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro methylation assay with mutagenesis, enzymatic activity assay, and localization experiments, single lab but multiple orthogonal methods","pmids":["29471495"],"is_preprint":false},{"year":2020,"finding":"TOP3B forms covalent cleavage complexes (TOP3Bccs) on both RNA and DNA substrates in vivo. A self-trapping mutant (R338W-TOP3B) induces R-loops and genomic damage. TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 prior to proteasomal processing. TDP2 (tyrosyl-DNA phosphodiesterase 2) repairs TOP3Bccs on both DNA and RNA substrates in vitro; TDP2-deficient cells show elevated TOP3Bccs, and TDP2 overexpression lowers cellular TOP3Bccs.","method":"Self-trapping mutant engineering, immunodetection of TOP3Bccs, TDP1/TDP2 depletion, overexpression, recombinant TDP2 in vitro assay, ubiquitination assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution with recombinant proteins, genetic depletion/overexpression with functional readouts, multiple orthogonal methods in single study","pmids":["33378676"],"is_preprint":false},{"year":2022,"finding":"TOP3B physically associates with R-loops and cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex, as shown by biochemical assays with recombinant TOP3B and oligonucleotides mimicking R-loops. TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel to senataxin. TOP3B knockout cells show elevated R-loops that are suppressed by TOP3B transfection.","method":"TOP3B knockout cells, R-loop detection (DRIP-seq), recombinant protein biochemical assay with R-loop mimics, IP-mass spectrometry, IP-western, epistasis analysis (double knockdown)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution with R-loop mimics, IP-MS, genetic epistasis (double KO/KD), knockout rescue, multiple orthogonal methods","pmids":["35830799"],"is_preprint":false},{"year":2019,"finding":"Loss of TOP3B in human cells causes increased R-loop formation, elevated DNA damage, and chromosome bridging/mis-segregation, establishing that TOP3B is necessary to prevent excessive R-loop accumulation and maintain genome stability.","method":"TOP3B deletion patient cells and modeled human cells, R-loop detection, γH2AX assay, chromosome bridge quantification","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in patient and modeled cells with specific cellular phenotypic readouts; single lab, multiple assays","pmids":["31795919"],"is_preprint":false},{"year":2023,"finding":"TOP3B protein stability is regulated by a balance between ubiquitylation and deubiquitylation: MIB1 E3 ligase directly ubiquitylates TOP3B (independently of TDRD3) to promote proteasomal degradation, while TDRD3 recruits the deubiquitinase USP9X to stabilize TOP3B. Absence of TDRD3 increases TOP3Bccs on DNA and RNA, R-loops, γH2AX, and growth defects. TDRD3 also directly increases TOP3B catalytic turnover in biochemical experiments.","method":"Co-IP (TOP3B-USP9X-TDRD3 interactions), USP9X inactivation, MIB1 knockdown, TOP3Bcc detection, R-loop detection, γH2AX assay, in vitro biochemical turnover assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, genetic depletion epistasis, TOP3Bcc and R-loop quantification, and in vitro biochemical assay; single lab, multiple orthogonal methods","pmids":["37980342"],"is_preprint":false},{"year":2025,"finding":"The autism-linked C666R mutation in TOP3B disrupts metal coordination within an atypical D1C3-type zinc finger domain, causing accumulation of unresolved TOP3B•mRNA covalent intermediates in cells. TOP3B•mRNA adducts cause ribosome collisions in primary neurons, identifying a previously unrecognized role of the zinc finger domain in TOP3B RNA activity.","method":"Cell-based TOP3B activity assay (Neuro2A), site-directed mutagenesis (C666R), oligo-dT pulldown of TOP3B•mRNA covalent intermediates, ribosome collision assay in primary neurons, biochemical metal coordination analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and biochemical evidence in cell-based and in vitro assays, single lab preprint, multiple orthogonal methods","pmids":[],"is_preprint":true},{"year":2025,"finding":"TOP3B forms covalent intermediates with mRNA (TOP3B•mRNA) that can be selectively captured from mammalian cells using oligo-dT pulldown under denaturing conditions, confirming catalytic activity of TOP3B on mRNA substrates in cells.","method":"Oligo-dT pulldown under denaturing conditions, dual-membrane slot blotting, immunodetection of TOP3B•mRNA covalent intermediates in Neuro2A cells","journal":"Bio-protocol","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct biochemical demonstration of TOP3B•mRNA covalent intermediates in cells with validated protocol, single lab, single method type","pmids":["41815835"],"is_preprint":false}],"current_model":"TOP3B is a dual DNA/RNA type IA topoisomerase that resolves topological stress and R-loops during transcription and translation: it forms a complex with TDRD3 (which bridges it to methylated chromatin and stabilizes it via USP9X recruitment) and with DDX5 (independently of TDRD3) to decatenate R-loops; its activity is positively regulated by PRMT1/3/6-mediated arginine methylation at its C-terminal RGG motif (R833/R835); it forms covalent cleavage complexes (TOP3Bccs) on both DNA and RNA that are repaired by TRIM41-mediated ubiquitination followed by TDP2-mediated excision; its zinc finger domain (with a D1C3-type metal binding motif) is required for catalytic resolution of TOP3B•mRNA intermediates; and its protein stability is governed by MIB1-mediated ubiquitination/proteasomal degradation counteracted by the TDRD3-USP9X deubiquitinase complex."},"narrative":{"mechanistic_narrative":"TOP3B is a dual DNA/RNA type IA topoisomerase that prevents excessive R-loop accumulation and maintains genome stability during transcription [PMID:24507716, PMID:31795919]. It is recruited to chromatin through a complex with TDRD3, which bridges TOP3B to arginine-methylated histones at gene promoters such as c-MYC, where TOP3B relaxes negatively supercoiled DNA and reduces transcription-generated R-loops; loss of this activity elevates R-loops, DNA damage, and chromosomal translocations [PMID:24507716, PMID:31795919]. TOP3B physically associates with R-loops and cleaves the displaced single-stranded DNA, acting together with the helicase DDX5 in a pathway parallel to senataxin, and it engages DDX5 independently of TDRD3 [PMID:35830799]. Its catalytic activity is positively regulated by PRMT1/3/6-mediated arginine methylation at R833/R835 in its C-terminal RGG motif, which also promotes TDRD3 binding and stress-granule localization [PMID:29471495]. In the course of catalysis TOP3B forms covalent cleavage complexes (TOP3Bccs) on both DNA and RNA, including covalent TOP3B•mRNA intermediates that can be captured from cells; these abortive adducts are resolved by TRIM41-mediated ubiquitination followed by TDP2-mediated excision [PMID:33378676, PMID:41815835]. An atypical D1C3-type zinc finger domain is required for resolving TOP3B•mRNA intermediates, and the autism-linked C666R mutation disrupts its metal coordination, causing accumulation of unresolved adducts and ribosome collisions in neurons. TOP3B abundance is set by a ubiquitylation balance: MIB1 ubiquitylates TOP3B to drive proteasomal degradation, while TDRD3 recruits the deubiquitinase USP9X to stabilize it and additionally enhances TOP3B catalytic turnover [PMID:37980342].","teleology":[{"year":2014,"claim":"Established that TOP3B is targeted to active gene loci and functions to suppress transcription-associated R-loops, answering how a topoisomerase is recruited to chromatin and what genome-protective role it serves.","evidence":"Co-IP, in vitro topoisomerase assay, DRIP/ChIP, and Tdrd3 knockout mouse with translocation readouts","pmids":["24507716"],"confidence":"High","gaps":["Did not define how TOP3B activity is regulated at the enzyme level","Did not establish whether TOP3B acts on RNA substrates directly"]},{"year":2018,"claim":"Identified post-translational control of TOP3B activity, showing arginine methylation of the C-terminal RGG motif tunes catalytic relaxation and partner binding.","evidence":"In vitro PRMT methylation assay, R833/835K mutagenesis, methylation-specific antibody, topoisomerase and R-loop assays, stress-granule immunofluorescence","pmids":["29471495"],"confidence":"High","gaps":["Stoichiometry and cellular dynamics of methylation not quantified","Functional consequence of stress-granule localization not resolved"]},{"year":2019,"claim":"Demonstrated through loss-of-function that TOP3B is necessary to restrain R-loops and preserve chromosome segregation, linking its activity to genome instability phenotypes.","evidence":"TOP3B deletion patient and modeled human cells with R-loop, γH2AX, and chromosome-bridge readouts","pmids":["31795919"],"confidence":"Medium","gaps":["Single-lab cellular study","Direct molecular mechanism of R-loop resolution not addressed here"]},{"year":2020,"claim":"Revealed that TOP3B forms abortive covalent cleavage complexes on both DNA and RNA and defined a repair route, answering how trapped topoisomerase adducts are cleared.","evidence":"R338W self-trapping mutant, TOP3Bcc immunodetection, TRIM41 ubiquitination assay, TDP1/TDP2 depletion and recombinant TDP2 in vitro repair assay","pmids":["33378676"],"confidence":"High","gaps":["Order and coupling of TRIM41 ubiquitination versus TDP2 excision not fully resolved","In vivo contribution of each repair arm to RNA versus DNA adducts not separated"]},{"year":2022,"claim":"Defined the biochemical mode of R-loop resolution and a parallel helicase pathway, showing TOP3B cleaves R-loop-displaced ssDNA and acts with DDX5 independently of TDRD3.","evidence":"Recombinant TOP3B with R-loop mimics, TOP3B knockout cells with DRIP-seq and rescue, IP-MS/IP-western, double-knockdown epistasis against senataxin","pmids":["35830799"],"confidence":"High","gaps":["How TOP3B and DDX5 are mechanistically coordinated on R-loops not detailed","Relative genomic distribution of the DDX5 versus senataxin pathways unresolved"]},{"year":2023,"claim":"Resolved how TOP3B abundance is controlled, identifying opposing MIB1 ubiquitylation and TDRD3-USP9X deubiquitylation that govern stability and a direct TDRD3 enhancement of turnover.","evidence":"Reciprocal Co-IP, MIB1 knockdown, USP9X inactivation, TOP3Bcc/R-loop/γH2AX quantification, and in vitro biochemical turnover assay","pmids":["37980342"],"confidence":"High","gaps":["Signals triggering MIB1-driven degradation not identified","Whether stability control and methylation regulation intersect not tested"]},{"year":2025,"claim":"Assigned a catalytic role to the atypical D1C3 zinc finger domain in resolving TOP3B•mRNA intermediates and linked a disease mutation to ribosome collisions, extending TOP3B function into translation.","evidence":"Neuro2A cell-based activity assay, C666R mutagenesis, oligo-dT pulldown of covalent intermediates, ribosome collision assay in primary neurons, metal coordination analysis (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Structural basis of zinc finger participation in catalysis not solved","Causal chain from mRNA adducts to neurodevelopmental phenotype not established"]},{"year":2025,"claim":"Provided a validated cellular assay confirming TOP3B forms covalent intermediates directly on mRNA, cementing mRNA as a bona fide in-cell substrate.","evidence":"Oligo-dT pulldown under denaturing conditions with dual-membrane slot blotting in Neuro2A cells","pmids":["41815835"],"confidence":"Medium","gaps":["Single method type","Identity of physiological mRNA targets not enumerated"]},{"year":null,"claim":"How TOP3B selects among DNA, R-loop, and mRNA substrates in different cellular contexts, and how its regulatory inputs (methylation, stability, zinc finger) are integrated, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating zinc finger, RGG motif, and catalytic core","Determinants of DNA-versus-RNA substrate choice in vivo unknown","Tissue-specific consequences of TOP3B dysfunction not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0,3]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[2,6,7]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,3]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2,7]},{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[2,4]}],"complexes":["TOP3B-TDRD3 complex"],"partners":["TDRD3","DDX5","USP9X","MIB1","TRIM41","TDP2","PRMT1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95985","full_name":"DNA topoisomerase 3-beta-1","aliases":["DNA topoisomerase III beta-1"],"length_aa":862,"mass_kda":96.7,"function":"Releases the supercoiling and torsional tension of DNA introduced during the DNA replication and transcription by transiently cleaving and rejoining one strand of the DNA duplex. Introduces a single-strand break via transesterification at a target site in duplex DNA. The scissile phosphodiester is attacked by the catalytic tyrosine of the enzyme, resulting in the formation of a DNA-(5'-phosphotyrosyl)-enzyme intermediate and the expulsion of a 3'-OH DNA strand. The free DNA strand than undergoes passage around the unbroken strand thus removing DNA supercoils. Finally, in the religation step, the DNA 3'-OH attacks the covalent intermediate to expel the active-site tyrosine and restore the DNA phosphodiester backbone (By similarity). Possesses negatively supercoiled DNA relaxing activity","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/O95985/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TOP3B","classification":"Not Classified","n_dependent_lines":21,"n_total_lines":1208,"dependency_fraction":0.0173841059602649},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ACTN1","stoichiometry":0.2},{"gene":"RBM8A","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2},{"gene":"SRP14","stoichiometry":0.2},{"gene":"SRP72","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TOP3B","total_profiled":1310},"omim":[{"mim_id":"611867","title":"CHROMOSOME 22q11.2 DELETION SYNDROME, DISTAL","url":"https://www.omim.org/entry/611867"},{"mim_id":"610530","title":"TRIPARTITE MOTIF-CONTAINING PROTEIN 41; TRIM41","url":"https://www.omim.org/entry/610530"},{"mim_id":"603582","title":"TOPOISOMERASE, DNA, III, BETA; TOP3B","url":"https://www.omim.org/entry/603582"},{"mim_id":"181500","title":"SCHIZOPHRENIA; SCZD","url":"https://www.omim.org/entry/181500"}],"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/TOP3B"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O95985","domains":[{"cath_id":"3.40.50.140","chopping":"1-58_93-170","consensus_level":"high","plddt":95.4185,"start":1,"end":170},{"cath_id":"1.10.460.10","chopping":"178-234_494-610","consensus_level":"high","plddt":94.0701,"start":178,"end":610},{"cath_id":"2.70.20.10","chopping":"238-295_424-486","consensus_level":"medium","plddt":96.0749,"start":238,"end":486},{"cath_id":"1.10.290.10","chopping":"302-421","consensus_level":"medium","plddt":96.8191,"start":302,"end":421},{"cath_id":"-","chopping":"721-818","consensus_level":"medium","plddt":85.3692,"start":721,"end":818},{"cath_id":"2.40.10","chopping":"613-712","consensus_level":"medium","plddt":87.4461,"start":613,"end":712}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95985","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95985-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95985-F1-predicted_aligned_error_v6.png","plddt_mean":90.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TOP3B","jax_strain_url":"https://www.jax.org/strain/search?query=TOP3B"},"sequence":{"accession":"O95985","fasta_url":"https://rest.uniprot.org/uniprotkb/O95985.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95985/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95985"}},"corpus_meta":[{"pmid":"24507716","id":"PMC_24507716","title":"Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/24507716","citation_count":201,"is_preprint":false},{"pmid":"29471495","id":"PMC_29471495","title":"Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization.","date":"2018","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/29471495","citation_count":73,"is_preprint":false},{"pmid":"35830799","id":"PMC_35830799","title":"Resolution of R-loops by topoisomerase III-β (TOP3B) in coordination with the DEAD-box helicase DDX5.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/35830799","citation_count":61,"is_preprint":false},{"pmid":"31795919","id":"PMC_31795919","title":"Loss of TOP3B leads to increased R-loop formation and genome instability.","date":"2019","source":"Open biology","url":"https://pubmed.ncbi.nlm.nih.gov/31795919","citation_count":53,"is_preprint":false},{"pmid":"33378676","id":"PMC_33378676","title":"DNA and RNA Cleavage Complexes and Repair Pathway for TOP3B RNA- and DNA-Protein Crosslinks.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/33378676","citation_count":46,"is_preprint":false},{"pmid":"27880953","id":"PMC_27880953","title":"Deletion of TOP3B Is Associated with Cognitive Impairment and Facial Dysmorphism.","date":"2016","source":"Cytogenetic and genome research","url":"https://pubmed.ncbi.nlm.nih.gov/27880953","citation_count":33,"is_preprint":false},{"pmid":"32028044","id":"PMC_32028044","title":"Further evidence of GABRA4 and TOP3B as autism susceptibility genes.","date":"2020","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32028044","citation_count":19,"is_preprint":false},{"pmid":"29490292","id":"PMC_29490292","title":"TOP3B: A Novel Candidate Gene in Juvenile Myoclonic Epilepsy?","date":"2018","source":"Cytogenetic and genome research","url":"https://pubmed.ncbi.nlm.nih.gov/29490292","citation_count":18,"is_preprint":false},{"pmid":"37980342","id":"PMC_37980342","title":"The TDRD3-USP9X complex and MIB1 regulate TOP3B homeostasis and prevent deleterious TOP3B cleavage complexes.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37980342","citation_count":15,"is_preprint":false},{"pmid":"33999213","id":"PMC_33999213","title":"Molecular Evolution of DNA Topoisomerase III Beta (TOP3B) in Metazoa.","date":"2021","source":"Journal of molecular evolution","url":"https://pubmed.ncbi.nlm.nih.gov/33999213","citation_count":9,"is_preprint":false},{"pmid":"40754336","id":"PMC_40754336","title":"Unraveling the Role of Topoisomerase 3β (TOP3B) in mRNA Translation and Human Disease.","date":"2025","source":"Wiley interdisciplinary reviews. RNA","url":"https://pubmed.ncbi.nlm.nih.gov/40754336","citation_count":2,"is_preprint":false},{"pmid":"40070068","id":"PMC_40070068","title":"In-silico Comparative Analysis of TOP3B Protein Mammals Species with Emphasis on Heterocephalus glaber and Homo sapiens.","date":"2025","source":"Current aging science","url":"https://pubmed.ncbi.nlm.nih.gov/40070068","citation_count":0,"is_preprint":false},{"pmid":"41815835","id":"PMC_41815835","title":"Selective Isolation of TOP3B•mRNA Covalent Intermediates Using Denaturing Oligo-dT Pulldown.","date":"2026","source":"Bio-protocol","url":"https://pubmed.ncbi.nlm.nih.gov/41815835","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.11.647616","title":"An autism spectrum disorder mutation in Topoisomerase 3β causes accumulation of covalent mRNA intermediates by disrupting metal binding within the zinc finger domain","date":"2025-04-11","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.11.647616","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.13.643061","title":"Topoisomerase IIIβ protects from tumorigenesis and immune dysregulation","date":"2025-03-15","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.13.643061","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.18.642440","title":"Identification of novel human topoisomerase III beta inhibitors","date":"2025-03-18","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.18.642440","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.09.06.611624","title":"Uukuniemi virus infection causes a pervasive remodelling of the RNA-binding proteome in tick cells","date":"2024-09-06","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.06.611624","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.06.17.599352","title":"Genome-wide Mapping of Topoisomerase Binding Sites Suggests Topoisomerase 3α (TOP3A) as a Reader of Transcription-Replication Conflicts (TRC)","date":"2024-06-21","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.17.599352","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10165,"output_tokens":2503,"usd":0.03402,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9702,"output_tokens":3332,"usd":0.065905,"stage2_stop_reason":"end_turn"},"total_usd":0.099925,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"TOP3B forms a complex with TDRD3, which acts as a molecular bridge between TOP3B and arginine-methylated histones (H4R3me2a). The TDRD3-TOP3B complex is recruited to the c-MYC gene promoter, where TOP3B relaxes negatively supercoiled DNA and reduces transcription-generated R-loops in vitro. TDRD3 knockdown in cells increases R-loop formation at the c-MYC locus, and Tdrd3 null mice show elevated R-loop formation and increased c-Myc/Igh chromosomal translocations.\",\n      \"method\": \"Co-immunoprecipitation, in vitro topoisomerase assay, R-loop detection (DRIP), ChIP, Tdrd3 knockout mouse model\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro biochemical assays, genetic knockout mouse model with multiple orthogonal readouts across labs\",\n      \"pmids\": [\"24507716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TOP3B is arginine-methylated at R833 and R835 in its C-terminal RGG motif by PRMT1, PRMT3, and PRMT6 in vitro. Methylation-deficient TOP3B (R833/835K) shows reduced relaxation of negatively supercoiled DNA and increased R-loop accumulation in vitro and in cells. Additionally, methylation at R833/835 is partially required for TOP3B interaction with TDRD3 and for TOP3B localization to stress granules.\",\n      \"method\": \"In vitro methylation assay, site-directed mutagenesis, methylation-specific antibody, in vitro topoisomerase assay, R-loop detection, immunofluorescence for stress granule localization, Co-IP\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro methylation assay with mutagenesis, enzymatic activity assay, and localization experiments, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"29471495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TOP3B forms covalent cleavage complexes (TOP3Bccs) on both RNA and DNA substrates in vivo. A self-trapping mutant (R338W-TOP3B) induces R-loops and genomic damage. TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 prior to proteasomal processing. TDP2 (tyrosyl-DNA phosphodiesterase 2) repairs TOP3Bccs on both DNA and RNA substrates in vitro; TDP2-deficient cells show elevated TOP3Bccs, and TDP2 overexpression lowers cellular TOP3Bccs.\",\n      \"method\": \"Self-trapping mutant engineering, immunodetection of TOP3Bccs, TDP1/TDP2 depletion, overexpression, recombinant TDP2 in vitro assay, ubiquitination assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution with recombinant proteins, genetic depletion/overexpression with functional readouts, multiple orthogonal methods in single study\",\n      \"pmids\": [\"33378676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TOP3B physically associates with R-loops and cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex, as shown by biochemical assays with recombinant TOP3B and oligonucleotides mimicking R-loops. TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel to senataxin. TOP3B knockout cells show elevated R-loops that are suppressed by TOP3B transfection.\",\n      \"method\": \"TOP3B knockout cells, R-loop detection (DRIP-seq), recombinant protein biochemical assay with R-loop mimics, IP-mass spectrometry, IP-western, epistasis analysis (double knockdown)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution with R-loop mimics, IP-MS, genetic epistasis (double KO/KD), knockout rescue, multiple orthogonal methods\",\n      \"pmids\": [\"35830799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Loss of TOP3B in human cells causes increased R-loop formation, elevated DNA damage, and chromosome bridging/mis-segregation, establishing that TOP3B is necessary to prevent excessive R-loop accumulation and maintain genome stability.\",\n      \"method\": \"TOP3B deletion patient cells and modeled human cells, R-loop detection, γH2AX assay, chromosome bridge quantification\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in patient and modeled cells with specific cellular phenotypic readouts; single lab, multiple assays\",\n      \"pmids\": [\"31795919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TOP3B protein stability is regulated by a balance between ubiquitylation and deubiquitylation: MIB1 E3 ligase directly ubiquitylates TOP3B (independently of TDRD3) to promote proteasomal degradation, while TDRD3 recruits the deubiquitinase USP9X to stabilize TOP3B. Absence of TDRD3 increases TOP3Bccs on DNA and RNA, R-loops, γH2AX, and growth defects. TDRD3 also directly increases TOP3B catalytic turnover in biochemical experiments.\",\n      \"method\": \"Co-IP (TOP3B-USP9X-TDRD3 interactions), USP9X inactivation, MIB1 knockdown, TOP3Bcc detection, R-loop detection, γH2AX assay, in vitro biochemical turnover assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, genetic depletion epistasis, TOP3Bcc and R-loop quantification, and in vitro biochemical assay; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"37980342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The autism-linked C666R mutation in TOP3B disrupts metal coordination within an atypical D1C3-type zinc finger domain, causing accumulation of unresolved TOP3B•mRNA covalent intermediates in cells. TOP3B•mRNA adducts cause ribosome collisions in primary neurons, identifying a previously unrecognized role of the zinc finger domain in TOP3B RNA activity.\",\n      \"method\": \"Cell-based TOP3B activity assay (Neuro2A), site-directed mutagenesis (C666R), oligo-dT pulldown of TOP3B•mRNA covalent intermediates, ribosome collision assay in primary neurons, biochemical metal coordination analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and biochemical evidence in cell-based and in vitro assays, single lab preprint, multiple orthogonal methods\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TOP3B forms covalent intermediates with mRNA (TOP3B•mRNA) that can be selectively captured from mammalian cells using oligo-dT pulldown under denaturing conditions, confirming catalytic activity of TOP3B on mRNA substrates in cells.\",\n      \"method\": \"Oligo-dT pulldown under denaturing conditions, dual-membrane slot blotting, immunodetection of TOP3B•mRNA covalent intermediates in Neuro2A cells\",\n      \"journal\": \"Bio-protocol\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct biochemical demonstration of TOP3B•mRNA covalent intermediates in cells with validated protocol, single lab, single method type\",\n      \"pmids\": [\"41815835\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TOP3B is a dual DNA/RNA type IA topoisomerase that resolves topological stress and R-loops during transcription and translation: it forms a complex with TDRD3 (which bridges it to methylated chromatin and stabilizes it via USP9X recruitment) and with DDX5 (independently of TDRD3) to decatenate R-loops; its activity is positively regulated by PRMT1/3/6-mediated arginine methylation at its C-terminal RGG motif (R833/R835); it forms covalent cleavage complexes (TOP3Bccs) on both DNA and RNA that are repaired by TRIM41-mediated ubiquitination followed by TDP2-mediated excision; its zinc finger domain (with a D1C3-type metal binding motif) is required for catalytic resolution of TOP3B•mRNA intermediates; and its protein stability is governed by MIB1-mediated ubiquitination/proteasomal degradation counteracted by the TDRD3-USP9X deubiquitinase complex.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TOP3B is a dual DNA/RNA type IA topoisomerase that prevents excessive R-loop accumulation and maintains genome stability during transcription [#0, #4]. It is recruited to chromatin through a complex with TDRD3, which bridges TOP3B to arginine-methylated histones at gene promoters such as c-MYC, where TOP3B relaxes negatively supercoiled DNA and reduces transcription-generated R-loops; loss of this activity elevates R-loops, DNA damage, and chromosomal translocations [#0, #4]. TOP3B physically associates with R-loops and cleaves the displaced single-stranded DNA, acting together with the helicase DDX5 in a pathway parallel to senataxin, and it engages DDX5 independently of TDRD3 [#3]. Its catalytic activity is positively regulated by PRMT1/3/6-mediated arginine methylation at R833/R835 in its C-terminal RGG motif, which also promotes TDRD3 binding and stress-granule localization [#1]. In the course of catalysis TOP3B forms covalent cleavage complexes (TOP3Bccs) on both DNA and RNA, including covalent TOP3B\\u2022mRNA intermediates that can be captured from cells; these abortive adducts are resolved by TRIM41-mediated ubiquitination followed by TDP2-mediated excision [#2, #7]. An atypical D1C3-type zinc finger domain is required for resolving TOP3B\\u2022mRNA intermediates, and the autism-linked C666R mutation disrupts its metal coordination, causing accumulation of unresolved adducts and ribosome collisions in neurons [#6]. TOP3B abundance is set by a ubiquitylation balance: MIB1 ubiquitylates TOP3B to drive proteasomal degradation, while TDRD3 recruits the deubiquitinase USP9X to stabilize it and additionally enhances TOP3B catalytic turnover [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Established that TOP3B is targeted to active gene loci and functions to suppress transcription-associated R-loops, answering how a topoisomerase is recruited to chromatin and what genome-protective role it serves.\",\n      \"evidence\": \"Co-IP, in vitro topoisomerase assay, DRIP/ChIP, and Tdrd3 knockout mouse with translocation readouts\",\n      \"pmids\": [\"24507716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how TOP3B activity is regulated at the enzyme level\", \"Did not establish whether TOP3B acts on RNA substrates directly\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified post-translational control of TOP3B activity, showing arginine methylation of the C-terminal RGG motif tunes catalytic relaxation and partner binding.\",\n      \"evidence\": \"In vitro PRMT methylation assay, R833/835K mutagenesis, methylation-specific antibody, topoisomerase and R-loop assays, stress-granule immunofluorescence\",\n      \"pmids\": [\"29471495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and cellular dynamics of methylation not quantified\", \"Functional consequence of stress-granule localization not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated through loss-of-function that TOP3B is necessary to restrain R-loops and preserve chromosome segregation, linking its activity to genome instability phenotypes.\",\n      \"evidence\": \"TOP3B deletion patient and modeled human cells with R-loop, \\u03b3H2AX, and chromosome-bridge readouts\",\n      \"pmids\": [\"31795919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab cellular study\", \"Direct molecular mechanism of R-loop resolution not addressed here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed that TOP3B forms abortive covalent cleavage complexes on both DNA and RNA and defined a repair route, answering how trapped topoisomerase adducts are cleared.\",\n      \"evidence\": \"R338W self-trapping mutant, TOP3Bcc immunodetection, TRIM41 ubiquitination assay, TDP1/TDP2 depletion and recombinant TDP2 in vitro repair assay\",\n      \"pmids\": [\"33378676\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order and coupling of TRIM41 ubiquitination versus TDP2 excision not fully resolved\", \"In vivo contribution of each repair arm to RNA versus DNA adducts not separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the biochemical mode of R-loop resolution and a parallel helicase pathway, showing TOP3B cleaves R-loop-displaced ssDNA and acts with DDX5 independently of TDRD3.\",\n      \"evidence\": \"Recombinant TOP3B with R-loop mimics, TOP3B knockout cells with DRIP-seq and rescue, IP-MS/IP-western, double-knockdown epistasis against senataxin\",\n      \"pmids\": [\"35830799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TOP3B and DDX5 are mechanistically coordinated on R-loops not detailed\", \"Relative genomic distribution of the DDX5 versus senataxin pathways unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved how TOP3B abundance is controlled, identifying opposing MIB1 ubiquitylation and TDRD3-USP9X deubiquitylation that govern stability and a direct TDRD3 enhancement of turnover.\",\n      \"evidence\": \"Reciprocal Co-IP, MIB1 knockdown, USP9X inactivation, TOP3Bcc/R-loop/\\u03b3H2AX quantification, and in vitro biochemical turnover assay\",\n      \"pmids\": [\"37980342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signals triggering MIB1-driven degradation not identified\", \"Whether stability control and methylation regulation intersect not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Assigned a catalytic role to the atypical D1C3 zinc finger domain in resolving TOP3B\\u2022mRNA intermediates and linked a disease mutation to ribosome collisions, extending TOP3B function into translation.\",\n      \"evidence\": \"Neuro2A cell-based activity assay, C666R mutagenesis, oligo-dT pulldown of covalent intermediates, ribosome collision assay in primary neurons, metal coordination analysis (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Structural basis of zinc finger participation in catalysis not solved\", \"Causal chain from mRNA adducts to neurodevelopmental phenotype not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a validated cellular assay confirming TOP3B forms covalent intermediates directly on mRNA, cementing mRNA as a bona fide in-cell substrate.\",\n      \"evidence\": \"Oligo-dT pulldown under denaturing conditions with dual-membrane slot blotting in Neuro2A cells\",\n      \"pmids\": [\"41815835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method type\", \"Identity of physiological mRNA targets not enumerated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TOP3B selects among DNA, R-loop, and mRNA substrates in different cellular contexts, and how its regulatory inputs (methylation, stability, zinc finger) are integrated, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating zinc finger, RGG motif, and catalytic core\", \"Determinants of DNA-versus-RNA substrate choice in vivo unknown\", \"Tissue-specific consequences of TOP3B dysfunction not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [2, 6, 7]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"complexes\": [\"TOP3B-TDRD3 complex\"],\n    \"partners\": [\"TDRD3\", \"DDX5\", \"USP9X\", \"MIB1\", \"TRIM41\", \"TDP2\", \"PRMT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}