{"gene":"TOP3A","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1992,"finding":"Yeast TOP3 gene product (74-kDa protein) was purified and shown to be a single-strand-specific type IA DNA topoisomerase that partially relaxes negatively but not positively supercoiled DNA, forms a covalent protein-DNA complex linked to the 5' DNA phosphoryl group, and has strong preference for single-stranded DNA substrates.","method":"Protein purification, in vitro topoisomerase assay, protein-DNA covalent complex identification, sequencing of cleavage sites","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro biochemical reconstitution with purified protein, active-site characterization, multiple orthogonal assays in a single rigorous study","pmids":["1324925"],"is_preprint":false},{"year":1994,"finding":"Yeast Top3 physically interacts with the RecQ helicase homolog Sgs1; Sgs1 was identified in a two-hybrid screen for Top3-interacting proteins, and mutations in SGS1 suppress both the growth defect and increased genomic instability of top3 mutants, placing Sgs1 and Top3 in the same pathway.","method":"Two-hybrid screen (protein interaction), genetic epistasis/suppressor analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two-hybrid interaction plus genetic epistasis, independently replicated in multiple subsequent studies","pmids":["7969174"],"is_preprint":false},{"year":1996,"finding":"Human TOP3A (TOP3) encodes a DNA topoisomerase III that reduces supercoils in negatively supercoiled DNA; expression of the human cDNA in yeast top1 cells lacking endogenous topoisomerase I yielded supercoil-reducing activity in cell extracts. The gene is located at chromosome 17p11.2-12.","method":"cDNA cloning, heterologous expression in yeast, in vitro topoisomerase activity assay, FISH chromosomal mapping","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic activity assay with heterologous expression and functional complementation, single lab but multiple orthogonal methods","pmids":["8622991"],"is_preprint":false},{"year":2001,"finding":"The N-terminal 45 amino acids of Sgs1 (specifically residues 4–13) are required for interaction with Top3; missense mutations in this region abolish Top3 binding and abolish complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants.","method":"Deletion and missense mutagenesis, two-hybrid interaction assay, DNA damage sensitivity assay","journal":"Molecular genetics and genomics : MGG","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two-hybrid interaction mapping plus functional phenotypic readouts, single lab","pmids":["11523801"],"is_preprint":false},{"year":2002,"finding":"Top3 DNA topoisomerase catalytic activity (requiring the active-site Tyr-356) is required to repair MMS-induced DNA damage independently of Sgs1; a catalytic-dead TOP3(Y356F) allele fails to restore MMS sensitivity of sgs1-top3 double mutants to sgs1 single mutant levels, and TOP3 is epistatic to RAD52.","method":"Active-site mutagenesis (Y356F), DNA damage sensitivity assay, epistasis analysis","journal":"Genes & genetic systems","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — active-site mutagenesis with functional phenotypic readout and epistasis, single lab","pmids":["12036100"],"is_preprint":false},{"year":2003,"finding":"Sgs1 and its associated topoisomerase Top3 suppress crossovers during double-strand break repair by removing double Holliday junction intermediates from a crossover-producing repair pathway in mitotic yeast cells.","method":"Genetic epistasis analysis, physical monitoring of recombination intermediates, deletion of SGS1/SRS2","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic deletion combinations with direct molecular monitoring of recombination products, highly cited and replicated","pmids":["14622595"],"is_preprint":false},{"year":2005,"finding":"Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast; Rmi1 interacts directly with Top3 in a recombinant system; loss of either Rmi1 or Top3 compromises the partner's interaction with Sgs1; Rmi1 is a structure-specific DNA binding protein with preference for cruciform structures.","method":"Co-immunoprecipitation, recombinant protein interaction assay, DNA binding assay, genetic epistasis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, recombinant protein interaction, and DNA binding biochemistry combined with genetic epistasis in a single study","pmids":["15899853"],"is_preprint":false},{"year":2006,"finding":"The catalytic (decatenation) activity of Top3 (Tyr-356 residue) is not required for DNA damage checkpoint activation but is required for normal S-phase progression after DNA damage; overexpression of catalytic-dead TOP3(Y356F) causes persistence of X-shaped DNA molecules after MMS exposure.","method":"Dominant-negative allele overexpression (TOP3-Y356F), 2D gel electrophoresis of DNA intermediates, checkpoint assays, caffeine override experiment","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative catalytic mutant with molecular readouts of DNA intermediates and checkpoint activity, single lab","pmids":["16899506"],"is_preprint":false},{"year":2010,"finding":"Sgs1 and Top3 proteins together are sufficient to migrate and disentangle a double Holliday junction (dHJ) to produce exclusively non-crossover recombination products (dissolution); Rmi1 stimulates dHJ dissolution by stimulating DNA decatenation (removing last strand linkages) rather than affecting initial HJ migration rate.","method":"In vitro biochemical reconstitution with purified proteins, dHJ dissolution assay","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins, mechanistic dissection of individual subunit contributions, replicated across labs","pmids":["20935631"],"is_preprint":false},{"year":2010,"finding":"Top3-Rmi1 heterodimer stimulates DNA end resection by forming a complex with Sgs1, which unexpectedly stimulates Sgs1 DNA unwinding activity; Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs.","method":"Biochemical reconstitution of DNA end resection in vitro with purified Dna2, Sgs1, RPA, Top3-Rmi1, MRX","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — full in vitro reconstitution with purified components, multiple mechanistic dissections, published in Nature","pmids":["20811461"],"is_preprint":false},{"year":2012,"finding":"Sgs1, Top3, Rmi1, and RPA coordinate dsDNA decatenation through sequential passage of single strands; Sgs1 is required for dsDNA unwinding and has a structural role in strand passage; RPA stimulates Sgs1 unwinding and Top3 strand passage; Rmi1 stabilizes the open Top3-DNA covalent complex intermediate and slows DNA relaxation but stimulates decatenation.","method":"In vitro biochemical reconstitution with purified proteins, catenation/decatenation assays, mechanistic dissection of individual subunit roles","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — full biochemical reconstitution with purified proteins and mechanistic dissection of each subunit's role in a single rigorous study","pmids":["22885009"],"is_preprint":false},{"year":2013,"finding":"The N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix (residues 25–38) critical for binding Top3 and Rmi1; proline substitutions disrupting this helix impair Top3/Rmi1 binding in vitro and cause hypersensitivity to DNA damaging agents and increased genome rearrangements in vivo.","method":"NMR spectroscopy, in vitro binding assays, proline mutagenesis, DNA damage sensitivity assays, genome stability assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure combined with mutagenesis and in vitro binding assays plus functional in vivo phenotypes, single lab but orthogonal methods","pmids":["24038467"],"is_preprint":false},{"year":2013,"finding":"Top3 alone (unassisted by Sgs1 and Rmi1) can resolve hemicatenane-related template switch recombination intermediates (Rec-X structures) but not double Holliday junction intermediates; purified Top3 resolves a synthetic Rec-X but not a synthetic dHJ in vitro.","method":"In vitro assay with purified Top3, synthetic Rec-X and dHJ substrates, 2D gel electrophoresis of genomic DNA, genetic experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro assay with purified protein on defined substrates, combined with genetic in vivo experiments, single lab","pmids":["24100144"],"is_preprint":false},{"year":2015,"finding":"Top3 disrupts D loops (displacement loops formed during homologous recombination) through a mechanism that depends on Top3's catalytic activity; Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54 but not protein-free D loops or those mediated by bacterial RecA or human RAD51/RAD54; the human Topoisomerase IIIα-RMI1-RMI2 complex also dissolves D loops.","method":"In vitro D-loop dissolution assay with purified proteins, catalytic mutant analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical reconstitution with purified proteins, multiple substrate specificity controls, catalytic dependence demonstrated, replicated with human complex","pmids":["25699708"],"is_preprint":false},{"year":2015,"finding":"Top3-Rmi1 strand-passage (decatenase) activity is required for all known Sgs1 functions in meiotic recombination, including channeling joint molecules into crossover and noncrossover pathways and suppressing non-allelic recombination; additionally, Top3-Rmi1 has a distinct Sgs1-independent late function in resolving recombination-dependent chromosome entanglements to allow anaphase segregation.","method":"Genetic analysis in yeast meiosis, catalytic mutant analysis, physical monitoring of joint molecule intermediates, chromosome segregation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — comprehensive genetic and physical analysis of Top3-Rmi1 catalytic mutants during meiosis, multiple independent labs publishing concordant findings (three papers simultaneously)","pmids":["25699709","25699707"],"is_preprint":false},{"year":2016,"finding":"Sgs1, Top3, and Rmi1 are sumoylated by the Smc5/6 SUMO E3 complex upon generation of recombination structures; sumoylation promotes STR inter-subunit interactions and accumulation at DNA repair centers; reduced STR sumoylation leads to accumulation of recombination intermediates.","method":"Co-immunoprecipitation, sumoylation assays, genetic analysis, live-cell imaging of repair foci","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and sumoylation assays with functional readouts, single lab, multiple orthogonal methods","pmids":["27373152"],"is_preprint":false},{"year":2018,"finding":"Biallelic mutations in TOP3A substantially reduce cellular levels of TopIIIα (the human TOP3A protein), leading to elevated sister chromatid exchanges (SCEs), chromosome segregation defects, and genome instability consistent with impaired dissolution of DNA recombination/replication intermediates; clinical features of mitochondrial dysfunction are also evident, consistent with a mitochondrial DNA decatenation function of TopIIIα.","method":"Patient-derived cell lines with biallelic TOP3A mutations, SCE assay, Western blot (protein levels), chromosome segregation analysis","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient cell lines with multiple orthogonal functional assays (SCE, protein levels, segregation), single study","pmids":["30057030"],"is_preprint":false},{"year":2019,"finding":"Human PICH helicase and Topoisomerase 3α (TOP3A) combine to create high-density positive DNA supercoiling in vitro, analogous to a reverse-gyrase activity; PICH progressively extrudes hypernegatively supercoiled DNA loops that are relaxed by TOP3A, generating positive supercoiling proposed to facilitate sister-chromatid disjunction by Topoisomerase 2α.","method":"Single-molecule manipulation, in vitro biochemical reconstitution of supercoiling activity with purified PICH and TOP3A","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins and direct single-molecule measurement of supercoiling activity, single lab but rigorous biophysical methods","pmids":["30936532"],"is_preprint":false},{"year":2019,"finding":"FANCM depletion provokes ALT (alternative lengthening of telomeres) activity; FANCM-mediated attenuation of ALT requires its interaction with the BLM-TOP3A-RMI (BTR) complex but not the FA core complex, indicating that FANCM functions with the BTR complex to restrain ALT replication stress at telomeres.","method":"siRNA depletion, ALT biomarker assays, break-induced telomere synthesis assays, co-immunoprecipitation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protein interaction (Co-IP) combined with functional depletion experiments and multiple ALT readouts, single lab","pmids":["31138797"],"is_preprint":false},{"year":2021,"finding":"Smc5/6 co-localizes with Sgs1-Top3-Rmi1 (STR) at natural pausing sites (NPSs) on chromosomes, facilitates Top3 retention at these sites, and individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (reversed forks, double Holliday junctions, hemicatenanes), indicating Smc5/6 regulates Top3 DNA processing activities at replication termination sites.","method":"ChIP co-localization, genetic depletion, 2D gel electrophoresis of DNA intermediates, intra-allelic suppressor isolation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP co-localization with genetic and molecular phenotypic analysis, single lab, multiple orthogonal methods","pmids":["33833229"],"is_preprint":false},{"year":2022,"finding":"TOP3A overexpression in ALT cancer cells counters ATRX-mediated ALT inhibition; TOP3A knockdown disrupts the ALT phenotype in ATRX-wt cells; TOP3A is required for proper BLM localization and promotes ALT DNA synthesis.","method":"TOP3A overexpression and knockdown in ALT cancer cell lines, BLM localization imaging, ALT DNA synthesis assay, functional genomics","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function and loss-of-function experiments with multiple functional readouts, single lab","pmids":["35920001"],"is_preprint":false},{"year":2023,"finding":"TOP3A localizes to both the nucleus and mitochondria via two distinct isoforms; pathogenic TOP3A variants cause either a Bloom syndrome-like nuclear disorder or adult-onset mitochondrial disease depending on overall severity of the catalytic defect, with milder variants selectively impairing mitochondrial DNA maintenance.","method":"Patient cell lines with biallelic TOP3A variants, mtDNA maintenance assays, enzyme activity characterization, cellular fractionation/localization","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple patient families with comprehensive functional characterization of mtDNA maintenance and enzymatic activity, single study","pmids":["37013609"],"is_preprint":false},{"year":2024,"finding":"TOP3A is enriched at telomeres of ALT cancer cells (but not telomerase-positive cells), stabilizes the shelterin protein TERF2 in ALT cells, promotes enrichment of long non-coding TERRA at telomeres, and promotes generation of single-stranded telomeric C-strand (ssTeloC) DNA; TOP3A-DNA-protein crosslinks suppress TERRA enrichment and destabilize TERF2.","method":"ChIP/telomere enrichment assays, protein stability assays, TERRA assay, ssTeloC detection, TOP3A-DNA crosslink induction","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, multiple assays but single lab, not yet peer-reviewed","pmids":["39803571"],"is_preprint":true},{"year":2024,"finding":"Genome-wide mapping shows TOP3A binding is concentrated at promoters and 5'-regions of transcribed genes and is suppressed by DNA replication inhibition, suggesting TOP3A is recruited to sites of transcription-replication conflicts (TRCs).","method":"CUT&Tag genome-wide topoisomerase binding mapping, replication inhibition experiment","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single method (binding mapping), no direct functional validation of TRC resolution by TOP3A","pmids":["38948815"],"is_preprint":true},{"year":2025,"finding":"The mitochondrial isoform of TOP3A undergoes proteolytic cleavage by the mitochondrial processing peptidase, removing ~90 amino acids from the C-terminus; this cleavage enhances single-stranded DNA binding and decatenation activity, and uncouples the mitochondrial isoform from nuclear BTRR complex protein interactions, enabling autonomous function in mtDNA maintenance.","method":"Protein biochemistry (cleavage identification), mitochondrial processing peptidase assay, in vitro ssDNA binding and decatenation activity assays, mass spectrometry, subcellular fractionation","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct enzymatic cleavage identification, in vitro activity assays with purified proteins, multiple orthogonal biochemical methods in a single rigorous peer-reviewed study","pmids":["41189053"],"is_preprint":false},{"year":2025,"finding":"RAD54L2 physically interacts with BLM (component of the BLM-TOP3A-RMI1-RMI2/BTRR complex) and suppresses sister chromatid exchanges; RAD54L2 is important for recruitment of BLM to chromatin and requires an intact ATPase domain to promote non-crossover recombination.","method":"Proximity proteomics (BioID), co-immunoprecipitation, SCE assay, chromatin fractionation","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity proteomics plus reciprocal Co-IP and functional SCE assay, single lab; finding pertains to BLM interaction rather than TOP3A directly","pmids":["39870965"],"is_preprint":false}],"current_model":"TOP3A encodes a type IA (single-strand-specific) DNA topoisomerase that operates primarily as a subunit of the conserved BLM-TOP3A-RMI1-RMI2 (BTRR) complex: it decatenates double Holliday junctions and hemicatenane intermediates during homologous recombination (dissolution) to produce exclusively non-crossover products, disrupts D-loops in a catalysis-dependent manner, stimulates DNA end resection by stabilizing Sgs1/BLM unwinding, and cooperates with PICH to generate positive DNA supercoiling for sister-chromatid disjunction; a separately processed mitochondrial isoform — activated by mitochondrial processing peptidase cleavage — functions autonomously to decatenate hemicatenated mtDNA daughter molecules after replication, and loss-of-function mutations cause a Bloom syndrome-like nuclear genome instability disorder or adult-onset mitochondrial disease depending on the severity of the catalytic defect."},"narrative":{"mechanistic_narrative":"TOP3A encodes a single-strand-specific type IA DNA topoisomerase that resolves recombination and replication intermediates to preserve genome stability, acting predominantly as the catalytic subunit of the conserved RecQ-helicase complex (Sgs1/BLM-Top3/TOP3A-Rmi1, the STR/BTR complex) [PMID:1324925, PMID:7969174, PMID:15899853]. The enzyme relaxes negatively supercoiled DNA, prefers single-stranded substrates, and operates through a covalent 5'-phosphotyrosyl protein-DNA intermediate at its active-site tyrosine (Tyr-356 in yeast), whose catalytic activity is required for repair of DNA damage and for normal S-phase progression [PMID:1324925, PMID:12036100, PMID:16899506]. Within the STR/BTR complex it migrates and decatenates double Holliday junctions to yield exclusively non-crossover products (dissolution), with the helicase providing strand unwinding, Top3 performing strand passage, and Rmi1 stabilizing the open Top3-DNA covalent intermediate to stimulate the final decatenation step [PMID:20935631, PMID:22885009]; the complex also disrupts D-loops in a catalysis-dependent manner and stimulates DNA end resection by enhancing helicase unwinding [PMID:25699708, PMID:20811461]. Beyond junction dissolution, TOP3A cooperates with the PICH helicase to generate positive DNA supercoiling that facilitates sister-chromatid disjunction [PMID:30936532], and Top3-Rmi1 has a helicase-independent late role in resolving recombination-dependent entanglements for anaphase segregation [PMID:25699709, PMID:25699707]. Its activity is regulated by Smc5/6-dependent sumoylation that promotes inter-subunit interactions and accumulation at repair centers, and by Smc5/6-mediated retention at replication-pausing sites [PMID:27373152, PMID:33833229]. A distinct mitochondrial isoform is matured by mitochondrial processing peptidase cleavage of ~90 C-terminal residues, which enhances ssDNA binding and decatenation and uncouples it from BTRR interactions, enabling autonomous mtDNA maintenance [PMID:41189053]. Biallelic TOP3A mutations reduce protein levels and cause elevated sister chromatid exchanges, chromosome segregation defects, and genome instability, producing either a Bloom syndrome-like nuclear disorder or adult-onset mitochondrial disease depending on the severity of the catalytic defect [PMID:30057030, PMID:37013609].","teleology":[{"year":1992,"claim":"Established the fundamental enzymatic identity of the TOP3 product, defining it as a single-strand-specific type IA topoisomerase rather than a conventional supercoil relaxase.","evidence":"Protein purification and in vitro topoisomerase assays with covalent protein-DNA complex mapping in yeast","pmids":["1324925"],"confidence":"High","gaps":["Cellular substrates and pathway context unknown","No partner proteins identified at this stage"]},{"year":1994,"claim":"Placed Top3 in a functional pathway by identifying the RecQ helicase Sgs1 as a physical and genetic partner, framing the topoisomerase as part of a helicase-coupled genome-stability module.","evidence":"Two-hybrid screen and genetic suppressor/epistasis analysis in yeast","pmids":["7969174"],"confidence":"High","gaps":["Biochemical mechanism of the Sgs1-Top3 partnership not defined","Substrate processed by the pair unknown"]},{"year":1996,"claim":"Demonstrated that the human TOP3A ortholog is a functional supercoil-reducing topoisomerase, extending the yeast biology to humans.","evidence":"cDNA cloning, heterologous yeast expression, topoisomerase activity assay, FISH mapping","pmids":["8622991"],"confidence":"High","gaps":["Human complex partners not yet established","In vivo function in human cells uncharacterized"]},{"year":2001,"claim":"Mapped the molecular interface of the helicase-topoisomerase interaction to the Sgs1 N-terminus, linking complex assembly to DNA repair function.","evidence":"Deletion/missense mutagenesis with two-hybrid and DNA damage sensitivity assays","pmids":["11523801"],"confidence":"Medium","gaps":["Structural basis of binding not resolved","Mapped on Sgs1 side, not Top3 side"]},{"year":2002,"claim":"Showed that Top3 catalytic activity has an Sgs1-independent role in DNA damage repair, separating the topoisomerase's enzymatic contribution from helicase association.","evidence":"Active-site Y356F mutagenesis, DNA damage sensitivity, and epistasis analysis in yeast","pmids":["12036100"],"confidence":"Medium","gaps":["Specific intermediate processed independently of Sgs1 not identified","Single lab"]},{"year":2003,"claim":"Defined the cellular outcome of the Sgs1-Top3 pathway as crossover suppression during double-strand break repair via removal of double Holliday junctions.","evidence":"Genetic epistasis and physical monitoring of recombination intermediates in mitotic yeast","pmids":["14622595"],"confidence":"High","gaps":["Direct biochemical demonstration of dHJ resolution not yet shown","Role of additional subunits unaddressed"]},{"year":2005,"claim":"Identified Rmi1 as the third subunit and a structure-specific DNA-binding component, establishing the heterotrimeric STR architecture.","evidence":"Co-IP, recombinant interaction, DNA binding assay, and genetic epistasis in yeast","pmids":["15899853"],"confidence":"High","gaps":["Mechanistic contribution of Rmi1 to catalysis not yet defined"]},{"year":2010,"claim":"Reconstituted dissolution from purified components, proving Sgs1-Top3 alone migrate and disentangle a dHJ to give exclusively non-crossovers and assigning Rmi1 a decatenation-stimulating role.","evidence":"In vitro reconstitution with purified proteins and dHJ dissolution assay","pmids":["20935631"],"confidence":"High","gaps":["Stoichiometry and dynamics in vivo not addressed","Regulation of the reaction unknown"]},{"year":2010,"claim":"Revealed a second activity of the complex in DNA end resection, where Top3-Rmi1 stimulates Sgs1 unwinding and aids recruitment of the resection machinery.","evidence":"Full in vitro reconstitution of resection with purified Dna2, Sgs1, RPA, Top3-Rmi1, MRX","pmids":["20811461"],"confidence":"High","gaps":["In vivo contribution relative to other resection nucleases not quantified"]},{"year":2012,"claim":"Dissected the strand-passage decatenation mechanism, assigning sequential roles to each subunit including RPA stimulation and Rmi1 stabilization of the open Top3-DNA intermediate.","evidence":"In vitro reconstitution and catenation/decatenation assays with purified proteins","pmids":["22885009"],"confidence":"High","gaps":["Structural snapshots of the open intermediate not provided"]},{"year":2013,"claim":"Refined the assembly interface using NMR, showing a transient α-helix in the disordered Sgs1 N-terminus mediates Top3/Rmi1 binding and is required for genome stability.","evidence":"NMR, in vitro binding, proline mutagenesis, and in vivo genome stability assays","pmids":["24038467"],"confidence":"High","gaps":["Full complex structure not resolved"]},{"year":2013,"claim":"Distinguished substrate-specific activities, showing Top3 alone can resolve hemicatenane-like Rec-X intermediates but requires the helicase for dHJ processing.","evidence":"In vitro assays with purified Top3 on synthetic substrates plus 2D gels and genetics","pmids":["24100144"],"confidence":"Medium","gaps":["In vivo prevalence of Rec-X substrates uncertain","Single lab"]},{"year":2015,"claim":"Demonstrated catalysis-dependent D-loop disruption by Top3 and the human TOP3A-RMI1-RMI2 complex, broadening the complex's anti-recombinogenic repertoire.","evidence":"In vitro D-loop dissolution assays with purified yeast and human proteins and catalytic mutants","pmids":["25699708"],"confidence":"High","gaps":["Why disruption is specific to Rad51/Rad54-mediated D-loops mechanistically unexplained"]},{"year":2015,"claim":"Established that Top3-Rmi1 strand-passage activity underlies all Sgs1 meiotic functions and revealed a separate Sgs1-independent role in resolving entanglements for anaphase segregation.","evidence":"Meiotic genetics, catalytic mutants, joint-molecule monitoring, and segregation assays (concordant multi-lab)","pmids":["25699709","25699707"],"confidence":"High","gaps":["Molecular nature of the late segregation substrate not defined"]},{"year":2016,"claim":"Identified Smc5/6-dependent sumoylation as a regulatory layer promoting STR subunit interactions and accumulation at repair centers.","evidence":"Co-IP, sumoylation assays, genetics, and live-cell imaging of repair foci","pmids":["27373152"],"confidence":"Medium","gaps":["Specific sumoylated residues and their individual effects not fully mapped","Single lab"]},{"year":2018,"claim":"Linked human TOP3A loss-of-function to disease, showing biallelic mutations reduce protein levels and cause SCE elevation, segregation defects, and mitochondrial dysfunction.","evidence":"Patient-derived cells with SCE, Western blot, and segregation analysis","pmids":["30057030"],"confidence":"Medium","gaps":["Genotype-phenotype relationship across variant severity not yet resolved","Single study"]},{"year":2019,"claim":"Uncovered a reverse-gyrase-like activity in which PICH and TOP3A together generate positive supercoiling proposed to aid sister-chromatid disjunction.","evidence":"Single-molecule manipulation and in vitro reconstitution with purified PICH and TOP3A","pmids":["30936532"],"confidence":"High","gaps":["In vivo requirement for this activity not demonstrated"]},{"year":2019,"claim":"Connected the BTR complex to telomere maintenance, showing FANCM restrains ALT through its interaction with BLM-TOP3A-RMI.","evidence":"siRNA depletion, ALT biomarker and break-induced synthesis assays, Co-IP","pmids":["31138797"],"confidence":"Medium","gaps":["Direct enzymatic role of TOP3A at ALT telomeres not isolated","Single lab"]},{"year":2021,"claim":"Showed Smc5/6 spatially regulates Top3 by promoting its retention at natural pausing sites where it processes joint molecules at replication termination.","evidence":"ChIP co-localization, depletion, 2D gels, and suppressor isolation in yeast","pmids":["33833229"],"confidence":"Medium","gaps":["Mechanism of Smc5/6-mediated retention unresolved","Single lab"]},{"year":2022,"claim":"Established TOP3A as a driver of the ALT phenotype in cancer cells, required for BLM localization and ALT DNA synthesis and countering ATRX-mediated inhibition.","evidence":"Overexpression/knockdown in ALT cell lines with BLM imaging and ALT synthesis assays","pmids":["35920001"],"confidence":"Medium","gaps":["Whether TOP3A catalytic activity is required for ALT not separated from scaffolding","Single lab"]},{"year":2023,"claim":"Defined the dual nuclear/mitochondrial localization and a severity-dependent disease spectrum, with milder catalytic defects selectively impairing mtDNA maintenance.","evidence":"Patient cells with mtDNA maintenance assays, enzyme activity, and fractionation","pmids":["37013609"],"confidence":"Medium","gaps":["Quantitative catalytic thresholds for each clinical outcome not established","Single study"]},{"year":2024,"claim":"Detailed TOP3A telomere functions in ALT cells, including TERF2 stabilization and TERRA/ssTeloC promotion, with DNA-protein crosslinks reversing these effects.","evidence":"Telomere ChIP, protein stability, TERRA and ssTeloC assays, crosslink induction (preprint)","pmids":["39803571"],"confidence":"Low","gaps":["Preprint, not peer-reviewed","Single lab","Direct catalytic mechanism on telomeric substrates not reconstituted"]},{"year":2024,"claim":"Mapped genome-wide TOP3A occupancy to promoters and 5'-regions of transcribed genes, implicating it in transcription-replication conflict sites.","evidence":"CUT&Tag binding mapping with replication inhibition (preprint)","pmids":["38948815"],"confidence":"Low","gaps":["Preprint, not peer-reviewed","No functional validation of TRC resolution","Binding does not establish activity"]},{"year":2025,"claim":"Explained how the mitochondrial isoform is functionally specialized, showing mitochondrial processing peptidase cleavage enhances ssDNA binding and decatenation and uncouples it from the nuclear BTRR complex.","evidence":"Cleavage identification, MPP assay, in vitro ssDNA binding/decatenation, mass spectrometry, fractionation","pmids":["41189053"],"confidence":"High","gaps":["In vivo regulation of cleavage timing not addressed"]},{"year":2025,"claim":"Added RAD54L2 as a regulator of the BTR complex, promoting BLM chromatin recruitment and suppressing SCEs via its ATPase domain.","evidence":"BioID proximity proteomics, Co-IP, SCE assay, chromatin fractionation","pmids":["39870965"],"confidence":"Medium","gaps":["Interaction is with BLM rather than TOP3A directly","Single lab"]},{"year":null,"claim":"How TOP3A activity is partitioned and regulated between its dissolution, resection-stimulating, supercoiling, telomeric ALT, and transcription-replication conflict roles in human cells remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of the human BTRR complex on substrate","Catalytic requirement for ALT function not separated from scaffolding","TRC resolution role rests only on binding maps"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0,2,13]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,24]},{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[21]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[21,24]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[19,23]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[5,8,13]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[14,17]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[14]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[19]}],"complexes":["BLM-TOP3A-RMI1-RMI2 (BTR/BTRR)","Sgs1-Top3-Rmi1 (STR)"],"partners":["BLM","SGS1","RMI1","RMI2","PICH","FANCM","SMC5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13472","full_name":"DNA topoisomerase 3-alpha","aliases":["DNA topoisomerase III alpha"],"length_aa":1001,"mass_kda":112.4,"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. 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TOP3 gene product (74-kDa protein) was purified and shown to be a single-strand-specific type IA DNA topoisomerase that partially relaxes negatively but not positively supercoiled DNA, forms a covalent protein-DNA complex linked to the 5' DNA phosphoryl group, and has strong preference for single-stranded DNA substrates.\",\n      \"method\": \"Protein purification, in vitro topoisomerase assay, protein-DNA covalent complex identification, sequencing of cleavage sites\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro biochemical reconstitution with purified protein, active-site characterization, multiple orthogonal assays in a single rigorous study\",\n      \"pmids\": [\"1324925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Yeast Top3 physically interacts with the RecQ helicase homolog Sgs1; Sgs1 was identified in a two-hybrid screen for Top3-interacting proteins, and mutations in SGS1 suppress both the growth defect and increased genomic instability of top3 mutants, placing Sgs1 and Top3 in the same pathway.\",\n      \"method\": \"Two-hybrid screen (protein interaction), genetic epistasis/suppressor analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two-hybrid interaction plus genetic epistasis, independently replicated in multiple subsequent studies\",\n      \"pmids\": [\"7969174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human TOP3A (TOP3) encodes a DNA topoisomerase III that reduces supercoils in negatively supercoiled DNA; expression of the human cDNA in yeast top1 cells lacking endogenous topoisomerase I yielded supercoil-reducing activity in cell extracts. The gene is located at chromosome 17p11.2-12.\",\n      \"method\": \"cDNA cloning, heterologous expression in yeast, in vitro topoisomerase activity assay, FISH chromosomal mapping\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic activity assay with heterologous expression and functional complementation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"8622991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The N-terminal 45 amino acids of Sgs1 (specifically residues 4–13) are required for interaction with Top3; missense mutations in this region abolish Top3 binding and abolish complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants.\",\n      \"method\": \"Deletion and missense mutagenesis, two-hybrid interaction assay, DNA damage sensitivity assay\",\n      \"journal\": \"Molecular genetics and genomics : MGG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid interaction mapping plus functional phenotypic readouts, single lab\",\n      \"pmids\": [\"11523801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Top3 DNA topoisomerase catalytic activity (requiring the active-site Tyr-356) is required to repair MMS-induced DNA damage independently of Sgs1; a catalytic-dead TOP3(Y356F) allele fails to restore MMS sensitivity of sgs1-top3 double mutants to sgs1 single mutant levels, and TOP3 is epistatic to RAD52.\",\n      \"method\": \"Active-site mutagenesis (Y356F), DNA damage sensitivity assay, epistasis analysis\",\n      \"journal\": \"Genes & genetic systems\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — active-site mutagenesis with functional phenotypic readout and epistasis, single lab\",\n      \"pmids\": [\"12036100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Sgs1 and its associated topoisomerase Top3 suppress crossovers during double-strand break repair by removing double Holliday junction intermediates from a crossover-producing repair pathway in mitotic yeast cells.\",\n      \"method\": \"Genetic epistasis analysis, physical monitoring of recombination intermediates, deletion of SGS1/SRS2\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic deletion combinations with direct molecular monitoring of recombination products, highly cited and replicated\",\n      \"pmids\": [\"14622595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast; Rmi1 interacts directly with Top3 in a recombinant system; loss of either Rmi1 or Top3 compromises the partner's interaction with Sgs1; Rmi1 is a structure-specific DNA binding protein with preference for cruciform structures.\",\n      \"method\": \"Co-immunoprecipitation, recombinant protein interaction assay, DNA binding assay, genetic epistasis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, recombinant protein interaction, and DNA binding biochemistry combined with genetic epistasis in a single study\",\n      \"pmids\": [\"15899853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The catalytic (decatenation) activity of Top3 (Tyr-356 residue) is not required for DNA damage checkpoint activation but is required for normal S-phase progression after DNA damage; overexpression of catalytic-dead TOP3(Y356F) causes persistence of X-shaped DNA molecules after MMS exposure.\",\n      \"method\": \"Dominant-negative allele overexpression (TOP3-Y356F), 2D gel electrophoresis of DNA intermediates, checkpoint assays, caffeine override experiment\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative catalytic mutant with molecular readouts of DNA intermediates and checkpoint activity, single lab\",\n      \"pmids\": [\"16899506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Sgs1 and Top3 proteins together are sufficient to migrate and disentangle a double Holliday junction (dHJ) to produce exclusively non-crossover recombination products (dissolution); Rmi1 stimulates dHJ dissolution by stimulating DNA decatenation (removing last strand linkages) rather than affecting initial HJ migration rate.\",\n      \"method\": \"In vitro biochemical reconstitution with purified proteins, dHJ dissolution assay\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins, mechanistic dissection of individual subunit contributions, replicated across labs\",\n      \"pmids\": [\"20935631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Top3-Rmi1 heterodimer stimulates DNA end resection by forming a complex with Sgs1, which unexpectedly stimulates Sgs1 DNA unwinding activity; Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs.\",\n      \"method\": \"Biochemical reconstitution of DNA end resection in vitro with purified Dna2, Sgs1, RPA, Top3-Rmi1, MRX\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — full in vitro reconstitution with purified components, multiple mechanistic dissections, published in Nature\",\n      \"pmids\": [\"20811461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Sgs1, Top3, Rmi1, and RPA coordinate dsDNA decatenation through sequential passage of single strands; Sgs1 is required for dsDNA unwinding and has a structural role in strand passage; RPA stimulates Sgs1 unwinding and Top3 strand passage; Rmi1 stabilizes the open Top3-DNA covalent complex intermediate and slows DNA relaxation but stimulates decatenation.\",\n      \"method\": \"In vitro biochemical reconstitution with purified proteins, catenation/decatenation assays, mechanistic dissection of individual subunit roles\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — full biochemical reconstitution with purified proteins and mechanistic dissection of each subunit's role in a single rigorous study\",\n      \"pmids\": [\"22885009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix (residues 25–38) critical for binding Top3 and Rmi1; proline substitutions disrupting this helix impair Top3/Rmi1 binding in vitro and cause hypersensitivity to DNA damaging agents and increased genome rearrangements in vivo.\",\n      \"method\": \"NMR spectroscopy, in vitro binding assays, proline mutagenesis, DNA damage sensitivity assays, genome stability assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure combined with mutagenesis and in vitro binding assays plus functional in vivo phenotypes, single lab but orthogonal methods\",\n      \"pmids\": [\"24038467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Top3 alone (unassisted by Sgs1 and Rmi1) can resolve hemicatenane-related template switch recombination intermediates (Rec-X structures) but not double Holliday junction intermediates; purified Top3 resolves a synthetic Rec-X but not a synthetic dHJ in vitro.\",\n      \"method\": \"In vitro assay with purified Top3, synthetic Rec-X and dHJ substrates, 2D gel electrophoresis of genomic DNA, genetic experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro assay with purified protein on defined substrates, combined with genetic in vivo experiments, single lab\",\n      \"pmids\": [\"24100144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Top3 disrupts D loops (displacement loops formed during homologous recombination) through a mechanism that depends on Top3's catalytic activity; Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54 but not protein-free D loops or those mediated by bacterial RecA or human RAD51/RAD54; the human Topoisomerase IIIα-RMI1-RMI2 complex also dissolves D loops.\",\n      \"method\": \"In vitro D-loop dissolution assay with purified proteins, catalytic mutant analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical reconstitution with purified proteins, multiple substrate specificity controls, catalytic dependence demonstrated, replicated with human complex\",\n      \"pmids\": [\"25699708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Top3-Rmi1 strand-passage (decatenase) activity is required for all known Sgs1 functions in meiotic recombination, including channeling joint molecules into crossover and noncrossover pathways and suppressing non-allelic recombination; additionally, Top3-Rmi1 has a distinct Sgs1-independent late function in resolving recombination-dependent chromosome entanglements to allow anaphase segregation.\",\n      \"method\": \"Genetic analysis in yeast meiosis, catalytic mutant analysis, physical monitoring of joint molecule intermediates, chromosome segregation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — comprehensive genetic and physical analysis of Top3-Rmi1 catalytic mutants during meiosis, multiple independent labs publishing concordant findings (three papers simultaneously)\",\n      \"pmids\": [\"25699709\", \"25699707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Sgs1, Top3, and Rmi1 are sumoylated by the Smc5/6 SUMO E3 complex upon generation of recombination structures; sumoylation promotes STR inter-subunit interactions and accumulation at DNA repair centers; reduced STR sumoylation leads to accumulation of recombination intermediates.\",\n      \"method\": \"Co-immunoprecipitation, sumoylation assays, genetic analysis, live-cell imaging of repair foci\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and sumoylation assays with functional readouts, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"27373152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Biallelic mutations in TOP3A substantially reduce cellular levels of TopIIIα (the human TOP3A protein), leading to elevated sister chromatid exchanges (SCEs), chromosome segregation defects, and genome instability consistent with impaired dissolution of DNA recombination/replication intermediates; clinical features of mitochondrial dysfunction are also evident, consistent with a mitochondrial DNA decatenation function of TopIIIα.\",\n      \"method\": \"Patient-derived cell lines with biallelic TOP3A mutations, SCE assay, Western blot (protein levels), chromosome segregation analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient cell lines with multiple orthogonal functional assays (SCE, protein levels, segregation), single study\",\n      \"pmids\": [\"30057030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human PICH helicase and Topoisomerase 3α (TOP3A) combine to create high-density positive DNA supercoiling in vitro, analogous to a reverse-gyrase activity; PICH progressively extrudes hypernegatively supercoiled DNA loops that are relaxed by TOP3A, generating positive supercoiling proposed to facilitate sister-chromatid disjunction by Topoisomerase 2α.\",\n      \"method\": \"Single-molecule manipulation, in vitro biochemical reconstitution of supercoiling activity with purified PICH and TOP3A\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins and direct single-molecule measurement of supercoiling activity, single lab but rigorous biophysical methods\",\n      \"pmids\": [\"30936532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FANCM depletion provokes ALT (alternative lengthening of telomeres) activity; FANCM-mediated attenuation of ALT requires its interaction with the BLM-TOP3A-RMI (BTR) complex but not the FA core complex, indicating that FANCM functions with the BTR complex to restrain ALT replication stress at telomeres.\",\n      \"method\": \"siRNA depletion, ALT biomarker assays, break-induced telomere synthesis assays, co-immunoprecipitation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protein interaction (Co-IP) combined with functional depletion experiments and multiple ALT readouts, single lab\",\n      \"pmids\": [\"31138797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Smc5/6 co-localizes with Sgs1-Top3-Rmi1 (STR) at natural pausing sites (NPSs) on chromosomes, facilitates Top3 retention at these sites, and individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (reversed forks, double Holliday junctions, hemicatenanes), indicating Smc5/6 regulates Top3 DNA processing activities at replication termination sites.\",\n      \"method\": \"ChIP co-localization, genetic depletion, 2D gel electrophoresis of DNA intermediates, intra-allelic suppressor isolation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP co-localization with genetic and molecular phenotypic analysis, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33833229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TOP3A overexpression in ALT cancer cells counters ATRX-mediated ALT inhibition; TOP3A knockdown disrupts the ALT phenotype in ATRX-wt cells; TOP3A is required for proper BLM localization and promotes ALT DNA synthesis.\",\n      \"method\": \"TOP3A overexpression and knockdown in ALT cancer cell lines, BLM localization imaging, ALT DNA synthesis assay, functional genomics\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function and loss-of-function experiments with multiple functional readouts, single lab\",\n      \"pmids\": [\"35920001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TOP3A localizes to both the nucleus and mitochondria via two distinct isoforms; pathogenic TOP3A variants cause either a Bloom syndrome-like nuclear disorder or adult-onset mitochondrial disease depending on overall severity of the catalytic defect, with milder variants selectively impairing mitochondrial DNA maintenance.\",\n      \"method\": \"Patient cell lines with biallelic TOP3A variants, mtDNA maintenance assays, enzyme activity characterization, cellular fractionation/localization\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple patient families with comprehensive functional characterization of mtDNA maintenance and enzymatic activity, single study\",\n      \"pmids\": [\"37013609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TOP3A is enriched at telomeres of ALT cancer cells (but not telomerase-positive cells), stabilizes the shelterin protein TERF2 in ALT cells, promotes enrichment of long non-coding TERRA at telomeres, and promotes generation of single-stranded telomeric C-strand (ssTeloC) DNA; TOP3A-DNA-protein crosslinks suppress TERRA enrichment and destabilize TERF2.\",\n      \"method\": \"ChIP/telomere enrichment assays, protein stability assays, TERRA assay, ssTeloC detection, TOP3A-DNA crosslink induction\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, multiple assays but single lab, not yet peer-reviewed\",\n      \"pmids\": [\"39803571\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Genome-wide mapping shows TOP3A binding is concentrated at promoters and 5'-regions of transcribed genes and is suppressed by DNA replication inhibition, suggesting TOP3A is recruited to sites of transcription-replication conflicts (TRCs).\",\n      \"method\": \"CUT&Tag genome-wide topoisomerase binding mapping, replication inhibition experiment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single method (binding mapping), no direct functional validation of TRC resolution by TOP3A\",\n      \"pmids\": [\"38948815\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The mitochondrial isoform of TOP3A undergoes proteolytic cleavage by the mitochondrial processing peptidase, removing ~90 amino acids from the C-terminus; this cleavage enhances single-stranded DNA binding and decatenation activity, and uncouples the mitochondrial isoform from nuclear BTRR complex protein interactions, enabling autonomous function in mtDNA maintenance.\",\n      \"method\": \"Protein biochemistry (cleavage identification), mitochondrial processing peptidase assay, in vitro ssDNA binding and decatenation activity assays, mass spectrometry, subcellular fractionation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct enzymatic cleavage identification, in vitro activity assays with purified proteins, multiple orthogonal biochemical methods in a single rigorous peer-reviewed study\",\n      \"pmids\": [\"41189053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RAD54L2 physically interacts with BLM (component of the BLM-TOP3A-RMI1-RMI2/BTRR complex) and suppresses sister chromatid exchanges; RAD54L2 is important for recruitment of BLM to chromatin and requires an intact ATPase domain to promote non-crossover recombination.\",\n      \"method\": \"Proximity proteomics (BioID), co-immunoprecipitation, SCE assay, chromatin fractionation\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity proteomics plus reciprocal Co-IP and functional SCE assay, single lab; finding pertains to BLM interaction rather than TOP3A directly\",\n      \"pmids\": [\"39870965\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TOP3A encodes a type IA (single-strand-specific) DNA topoisomerase that operates primarily as a subunit of the conserved BLM-TOP3A-RMI1-RMI2 (BTRR) complex: it decatenates double Holliday junctions and hemicatenane intermediates during homologous recombination (dissolution) to produce exclusively non-crossover products, disrupts D-loops in a catalysis-dependent manner, stimulates DNA end resection by stabilizing Sgs1/BLM unwinding, and cooperates with PICH to generate positive DNA supercoiling for sister-chromatid disjunction; a separately processed mitochondrial isoform — activated by mitochondrial processing peptidase cleavage — functions autonomously to decatenate hemicatenated mtDNA daughter molecules after replication, and loss-of-function mutations cause a Bloom syndrome-like nuclear genome instability disorder or adult-onset mitochondrial disease depending on the severity of the catalytic defect.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TOP3A encodes a single-strand-specific type IA DNA topoisomerase that resolves recombination and replication intermediates to preserve genome stability, acting predominantly as the catalytic subunit of the conserved RecQ-helicase complex (Sgs1/BLM-Top3/TOP3A-Rmi1, the STR/BTR complex) [#0, #1, #6]. The enzyme relaxes negatively supercoiled DNA, prefers single-stranded substrates, and operates through a covalent 5'-phosphotyrosyl protein-DNA intermediate at its active-site tyrosine (Tyr-356 in yeast), whose catalytic activity is required for repair of DNA damage and for normal S-phase progression [#0, #4, #7]. Within the STR/BTR complex it migrates and decatenates double Holliday junctions to yield exclusively non-crossover products (dissolution), with the helicase providing strand unwinding, Top3 performing strand passage, and Rmi1 stabilizing the open Top3-DNA covalent intermediate to stimulate the final decatenation step [#8, #10]; the complex also disrupts D-loops in a catalysis-dependent manner and stimulates DNA end resection by enhancing helicase unwinding [#13, #9]. Beyond junction dissolution, TOP3A cooperates with the PICH helicase to generate positive DNA supercoiling that facilitates sister-chromatid disjunction [#17], and Top3-Rmi1 has a helicase-independent late role in resolving recombination-dependent entanglements for anaphase segregation [#14]. Its activity is regulated by Smc5/6-dependent sumoylation that promotes inter-subunit interactions and accumulation at repair centers, and by Smc5/6-mediated retention at replication-pausing sites [#15, #19]. A distinct mitochondrial isoform is matured by mitochondrial processing peptidase cleavage of ~90 C-terminal residues, which enhances ssDNA binding and decatenation and uncouples it from BTRR interactions, enabling autonomous mtDNA maintenance [#24]. Biallelic TOP3A mutations reduce protein levels and cause elevated sister chromatid exchanges, chromosome segregation defects, and genome instability, producing either a Bloom syndrome-like nuclear disorder or adult-onset mitochondrial disease depending on the severity of the catalytic defect [#16, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established the fundamental enzymatic identity of the TOP3 product, defining it as a single-strand-specific type IA topoisomerase rather than a conventional supercoil relaxase.\",\n      \"evidence\": \"Protein purification and in vitro topoisomerase assays with covalent protein-DNA complex mapping in yeast\",\n      \"pmids\": [\"1324925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular substrates and pathway context unknown\", \"No partner proteins identified at this stage\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Placed Top3 in a functional pathway by identifying the RecQ helicase Sgs1 as a physical and genetic partner, framing the topoisomerase as part of a helicase-coupled genome-stability module.\",\n      \"evidence\": \"Two-hybrid screen and genetic suppressor/epistasis analysis in yeast\",\n      \"pmids\": [\"7969174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism of the Sgs1-Top3 partnership not defined\", \"Substrate processed by the pair unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrated that the human TOP3A ortholog is a functional supercoil-reducing topoisomerase, extending the yeast biology to humans.\",\n      \"evidence\": \"cDNA cloning, heterologous yeast expression, topoisomerase activity assay, FISH mapping\",\n      \"pmids\": [\"8622991\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human complex partners not yet established\", \"In vivo function in human cells uncharacterized\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Mapped the molecular interface of the helicase-topoisomerase interaction to the Sgs1 N-terminus, linking complex assembly to DNA repair function.\",\n      \"evidence\": \"Deletion/missense mutagenesis with two-hybrid and DNA damage sensitivity assays\",\n      \"pmids\": [\"11523801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of binding not resolved\", \"Mapped on Sgs1 side, not Top3 side\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed that Top3 catalytic activity has an Sgs1-independent role in DNA damage repair, separating the topoisomerase's enzymatic contribution from helicase association.\",\n      \"evidence\": \"Active-site Y356F mutagenesis, DNA damage sensitivity, and epistasis analysis in yeast\",\n      \"pmids\": [\"12036100\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific intermediate processed independently of Sgs1 not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the cellular outcome of the Sgs1-Top3 pathway as crossover suppression during double-strand break repair via removal of double Holliday junctions.\",\n      \"evidence\": \"Genetic epistasis and physical monitoring of recombination intermediates in mitotic yeast\",\n      \"pmids\": [\"14622595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical demonstration of dHJ resolution not yet shown\", \"Role of additional subunits unaddressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified Rmi1 as the third subunit and a structure-specific DNA-binding component, establishing the heterotrimeric STR architecture.\",\n      \"evidence\": \"Co-IP, recombinant interaction, DNA binding assay, and genetic epistasis in yeast\",\n      \"pmids\": [\"15899853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic contribution of Rmi1 to catalysis not yet defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Reconstituted dissolution from purified components, proving Sgs1-Top3 alone migrate and disentangle a dHJ to give exclusively non-crossovers and assigning Rmi1 a decatenation-stimulating role.\",\n      \"evidence\": \"In vitro reconstitution with purified proteins and dHJ dissolution assay\",\n      \"pmids\": [\"20935631\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and dynamics in vivo not addressed\", \"Regulation of the reaction unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed a second activity of the complex in DNA end resection, where Top3-Rmi1 stimulates Sgs1 unwinding and aids recruitment of the resection machinery.\",\n      \"evidence\": \"Full in vitro reconstitution of resection with purified Dna2, Sgs1, RPA, Top3-Rmi1, MRX\",\n      \"pmids\": [\"20811461\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution relative to other resection nucleases not quantified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Dissected the strand-passage decatenation mechanism, assigning sequential roles to each subunit including RPA stimulation and Rmi1 stabilization of the open Top3-DNA intermediate.\",\n      \"evidence\": \"In vitro reconstitution and catenation/decatenation assays with purified proteins\",\n      \"pmids\": [\"22885009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural snapshots of the open intermediate not provided\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Refined the assembly interface using NMR, showing a transient α-helix in the disordered Sgs1 N-terminus mediates Top3/Rmi1 binding and is required for genome stability.\",\n      \"evidence\": \"NMR, in vitro binding, proline mutagenesis, and in vivo genome stability assays\",\n      \"pmids\": [\"24038467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full complex structure not resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Distinguished substrate-specific activities, showing Top3 alone can resolve hemicatenane-like Rec-X intermediates but requires the helicase for dHJ processing.\",\n      \"evidence\": \"In vitro assays with purified Top3 on synthetic substrates plus 2D gels and genetics\",\n      \"pmids\": [\"24100144\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo prevalence of Rec-X substrates uncertain\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated catalysis-dependent D-loop disruption by Top3 and the human TOP3A-RMI1-RMI2 complex, broadening the complex's anti-recombinogenic repertoire.\",\n      \"evidence\": \"In vitro D-loop dissolution assays with purified yeast and human proteins and catalytic mutants\",\n      \"pmids\": [\"25699708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why disruption is specific to Rad51/Rad54-mediated D-loops mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established that Top3-Rmi1 strand-passage activity underlies all Sgs1 meiotic functions and revealed a separate Sgs1-independent role in resolving entanglements for anaphase segregation.\",\n      \"evidence\": \"Meiotic genetics, catalytic mutants, joint-molecule monitoring, and segregation assays (concordant multi-lab)\",\n      \"pmids\": [\"25699709\", \"25699707\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular nature of the late segregation substrate not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified Smc5/6-dependent sumoylation as a regulatory layer promoting STR subunit interactions and accumulation at repair centers.\",\n      \"evidence\": \"Co-IP, sumoylation assays, genetics, and live-cell imaging of repair foci\",\n      \"pmids\": [\"27373152\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific sumoylated residues and their individual effects not fully mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked human TOP3A loss-of-function to disease, showing biallelic mutations reduce protein levels and cause SCE elevation, segregation defects, and mitochondrial dysfunction.\",\n      \"evidence\": \"Patient-derived cells with SCE, Western blot, and segregation analysis\",\n      \"pmids\": [\"30057030\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genotype-phenotype relationship across variant severity not yet resolved\", \"Single study\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Uncovered a reverse-gyrase-like activity in which PICH and TOP3A together generate positive supercoiling proposed to aid sister-chromatid disjunction.\",\n      \"evidence\": \"Single-molecule manipulation and in vitro reconstitution with purified PICH and TOP3A\",\n      \"pmids\": [\"30936532\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo requirement for this activity not demonstrated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected the BTR complex to telomere maintenance, showing FANCM restrains ALT through its interaction with BLM-TOP3A-RMI.\",\n      \"evidence\": \"siRNA depletion, ALT biomarker and break-induced synthesis assays, Co-IP\",\n      \"pmids\": [\"31138797\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic role of TOP3A at ALT telomeres not isolated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed Smc5/6 spatially regulates Top3 by promoting its retention at natural pausing sites where it processes joint molecules at replication termination.\",\n      \"evidence\": \"ChIP co-localization, depletion, 2D gels, and suppressor isolation in yeast\",\n      \"pmids\": [\"33833229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of Smc5/6-mediated retention unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established TOP3A as a driver of the ALT phenotype in cancer cells, required for BLM localization and ALT DNA synthesis and countering ATRX-mediated inhibition.\",\n      \"evidence\": \"Overexpression/knockdown in ALT cell lines with BLM imaging and ALT synthesis assays\",\n      \"pmids\": [\"35920001\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TOP3A catalytic activity is required for ALT not separated from scaffolding\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the dual nuclear/mitochondrial localization and a severity-dependent disease spectrum, with milder catalytic defects selectively impairing mtDNA maintenance.\",\n      \"evidence\": \"Patient cells with mtDNA maintenance assays, enzyme activity, and fractionation\",\n      \"pmids\": [\"37013609\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative catalytic thresholds for each clinical outcome not established\", \"Single study\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Detailed TOP3A telomere functions in ALT cells, including TERF2 stabilization and TERRA/ssTeloC promotion, with DNA-protein crosslinks reversing these effects.\",\n      \"evidence\": \"Telomere ChIP, protein stability, TERRA and ssTeloC assays, crosslink induction (preprint)\",\n      \"pmids\": [\"39803571\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Single lab\", \"Direct catalytic mechanism on telomeric substrates not reconstituted\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapped genome-wide TOP3A occupancy to promoters and 5'-regions of transcribed genes, implicating it in transcription-replication conflict sites.\",\n      \"evidence\": \"CUT&Tag binding mapping with replication inhibition (preprint)\",\n      \"pmids\": [\"38948815\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"No functional validation of TRC resolution\", \"Binding does not establish activity\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Explained how the mitochondrial isoform is functionally specialized, showing mitochondrial processing peptidase cleavage enhances ssDNA binding and decatenation and uncouples it from the nuclear BTRR complex.\",\n      \"evidence\": \"Cleavage identification, MPP assay, in vitro ssDNA binding/decatenation, mass spectrometry, fractionation\",\n      \"pmids\": [\"41189053\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo regulation of cleavage timing not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added RAD54L2 as a regulator of the BTR complex, promoting BLM chromatin recruitment and suppressing SCEs via its ATPase domain.\",\n      \"evidence\": \"BioID proximity proteomics, Co-IP, SCE assay, chromatin fractionation\",\n      \"pmids\": [\"39870965\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction is with BLM rather than TOP3A directly\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TOP3A activity is partitioned and regulated between its dissolution, resection-stimulating, supercoiling, telomeric ALT, and transcription-replication conflict roles in human cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of the human BTRR complex on substrate\", \"Catalytic requirement for ALT function not separated from scaffolding\", \"TRC resolution role rests only on binding maps\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0, 2, 13]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 24]},\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [21]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [21, 24]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [19, 23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [5, 8, 13]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [14, 17]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"complexes\": [\"BLM-TOP3A-RMI1-RMI2 (BTR/BTRR)\", \"Sgs1-Top3-Rmi1 (STR)\"],\n    \"partners\": [\"BLM\", \"SGS1\", \"RMI1\", \"RMI2\", \"PICH\", \"FANCM\", \"SMC5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}