{"gene":"TOP2A","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2007,"finding":"Top2 acts within a ~600 bp region spanning moving replication forks to counteract torsional stress; top2 single mutants accumulate sister chromatid junctions in S phase and activate the Rad53 checkpoint at M-G1 transition; top1 top2 double mutants exhibit fork block, DNA damage checkpoint activation, and chromosome breakage, showing coordinated action of Top1 and Top2 at replication forks.","method":"ChIP-on-chip mapping of Top1/Top2 on replicating yeast chromosomes; genetic analysis of top1, top2, and top1 top2 double mutants; checkpoint kinase (Rad53) phosphorylation assays; Exo1 epistasis experiments","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genomic mapping plus multi-gene epistasis, replicated across multiple mutant combinations in a single rigorous study","pmids":["17671091"],"is_preprint":false},{"year":2006,"finding":"Tyrosyl-DNA phosphodiesterase (Tdp1) participates in repair of Top2-mediated DNA damage: Tdp1 can remove a Top2-derived peptide covalently linked to DNA via a 5'-phosphotyrosyl bond in vitro; tdp1 deletion confers hypersensitivity to Top2-targeting drugs; Tdp1 acts in collaboration with NHEJ, excision repair, and post-replication repair pathways.","method":"Yeast genetic deletion analysis; in vitro biochemical assay with bacterially expressed Tdp1p acting on 5'-phosphotyrosyl-linked Top2 peptide-DNA substrate; drug sensitivity assays; double-mutant epistasis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of enzymatic activity plus genetic epistasis across multiple pathways","pmids":["16751265"],"is_preprint":false},{"year":2014,"finding":"Irreversible Top2-DNA covalent complexes (Top2cc) require proteolytic processing (proteasomal degradation or denaturation) before TDP2 can remove the remaining 5'-phosphotyrosyl adduct; TDP2 is most active when the tyrosyl-linked DNA is single-stranded; TDP2 can also process tyrosine linked to RNA substrates. A 1.6 Å crystal structure of TDP2 bound to a 5'-ribonucleotide-bearing substrate explains RNA accommodation in the active site.","method":"In vitro TOP2cc processing assays with suicidal substrates; TDP2 enzymatic assays with varied substrates; crystal structure at 1.6 Å resolution; protease/denaturation pre-treatment experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mechanistic substrate variants plus high-resolution crystal structure in a single study","pmids":["24808172"],"is_preprint":false},{"year":2009,"finding":"Top2 binds intergenic regions near transcribed genes specifically in S phase; Top2-bound loci exhibit low nucleosome density; loss of Top2 causes γH2A accumulation at these loci; HMG protein Hmo1 co-occupies these loci and is deleterious in top2 mutants. Top2 is dispensable for transcription per se but suppresses chromosome fragility at M-G1 transition at transcription-associated loci.","method":"ChIP-chip mapping of Top2 and Hmo1 across yeast cell cycle; γH2A ChIP; genetic analysis of top2 and hmo1 mutants; nucleosome occupancy assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide ChIP-chip plus genetic epistasis with multiple chromatin markers; published in high-impact journal with multiple orthogonal methods","pmids":["19737516"],"is_preprint":false},{"year":2009,"finding":"Drug-poisoned TOP2α undergoes proteasomal degradation; the E3 ubiquitin ligase Bmi1/Ring1A ubiquitinates TOP2α in vitro and in cells; siRNA silencing of Bmi1 inhibits drug-induced TOP2α degradation and increases TOP2α-DNA cleavage complex persistence and drug efficacy. A small-molecule inhibitor of Bmi1/Ring1A ubiquitination activity prevents TOP2α ubiquitination and drug-induced degradation, synergistically enhancing TOP2 poison efficacy.","method":"siRNA screen identifying Bmi1/Ring1A; in vitro ubiquitination assay; cellular overexpression of Bmi1; drug sensitivity assays; small-molecule inhibitor of ubiquitin ligase","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro ubiquitination reconstitution plus cellular siRNA and overexpression experiments with functional readouts, multiple orthogonal methods in a single study","pmids":["19956605"],"is_preprint":false},{"year":2015,"finding":"TOP2 (but not TOP1) synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin, independent of transcription; this indicates that DNA decatenation/catenation topology changes (not torsional swiveling) are required for heterochromatin resolution. TOP2 also plays a role in re-formation of facultative heterochromatin, suggesting heterochromatin and accessible chromatin differ in catenation states.","method":"Genome-wide ATAC-seq and chromatin accessibility assays; TOP2 and TOP1 inhibitor treatments; BAF complex genetic perturbations; in vivo chromatin remodeling assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genome-wide chromatin accessibility mapping with multiple pharmacological and genetic perturbations; mechanistic distinction between TOP1 and TOP2 functions established","pmids":["28250416"],"is_preprint":false},{"year":2019,"finding":"TOP2A cleavage activity in humans is distributed in two fractions: tightly localized CTCF-proximal sites and broadly distributed transcription-proximal sites (correlated with gene length and transcript abundance); single-nucleotide mapping distinguishes canonical DSB sites from strand-biased SSB-prone sites induced by etoposide; Mre11-dependent repair of Top2 breaks was characterized in yeast.","method":"Strand-specific nucleotide-resolution mapping of Top2 DNA cleavage (END-seq derivative) in S. cerevisiae and human genomes; comparison with ENCODE chromatin marks; etoposide treatment; meiotic Spo11 validation","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — nucleotide-resolution genome-wide mapping across two organisms with multiple biological validations and functional annotations","pmids":["31649282"],"is_preprint":false},{"year":2020,"finding":"Upon replication stress, TOP2A is recruited to stalled replication forks in a manner dependent on HLTF, ZRANB3, and SMARCAL1; TOP2A undergoes SUMOylation mediated by the SUMO E3 ligase ZATT; SUMOylated TOP2A then recruits the SUMO-targeted DNA translocase PICH; this ZATT-TOP2A-PICH axis drives extensive fork reversal by resolving topological barriers. Loss of this axis causes accumulation of partially reversed forks and genome instability.","method":"Co-immunoprecipitation; proximity ligation assays; siRNA knockdown of HLTF/ZRANB3/SMARCAL1/ZATT/PICH; fork reversal electron microscopy; SUMOylation assays; genome instability measurements","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple reciprocal co-IPs, SUMOylation assays, genetic knockdowns with electron microscopy phenotypic readout, multiple orthogonal methods in a single study","pmids":["33296677"],"is_preprint":false},{"year":2011,"finding":"HuR binds the TOP2A 3'-UTR and increases TOP2A translation; reducing HuR triggers recruitment of TOP2A mRNA to RISC components and cytoplasmic processing bodies; miR-548c-3p, identified by MS2-tagged RNA precipitation, mediates repression of TOP2A translation by antagonizing HuR; lowering TOP2A by HuR silencing or miR-548c-3p overexpression decreases DNA damage after doxorubicin treatment.","method":"RNA-binding protein immunoprecipitation; MS2-tagged RNA precipitation to identify miR-548c-3p; reporter assays; siRNA knockdown of HuR; miRNA overexpression; doxorubicin cytotoxicity assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal RNA-protein interaction methods plus functional validation with drug treatment in a single study","pmids":["21768308"],"is_preprint":false},{"year":2015,"finding":"PTEN physically associates with TOP2A and stabilizes it through the deubiquitinase OTUD3; in PTEN-deficient cells, TOP2A ubiquitination increases and TOP2A protein levels decrease, leading to defective DNA decatenation checkpoint in G2, accumulation of ultra-fine anaphase bridges, and incomplete DNA decatenation.","method":"Co-immunoprecipitation of PTEN and TOP2A; ubiquitination assays; OTUD3 knockdown; analysis of ultra-fine bridges; decatenation checkpoint assays in PTEN-null cells","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional cellular assays in a single lab, multiple readouts but no in vitro reconstitution","pmids":["26657567"],"is_preprint":false},{"year":2019,"finding":"MDM4 and TOP2A physically bind each other; the C-terminal region (CTR) of TOP2A binds residues 188–238 of MDM4; this interaction stabilizes TOP2A protein post-translationally, and TOP2A binding activates MDM4 for p53 binding, resulting in enhanced p53 inhibition and increased cancer cell proliferation.","method":"Co-immunoprecipitation; domain-mapping experiments with truncation constructs; siRNA knockdown; cell proliferation assays; p53 activity assays","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP with domain mapping in a single lab, functional validation by KD/OE but no in vitro reconstitution","pmids":["30672125"],"is_preprint":false},{"year":2017,"finding":"TOP2 activity and transcription both contribute to DNA double-strand break formation after G4 ligand (pyridostatin and CX-5461) treatment; TOP2A was identified as a major effector of cytotoxicity by an unbiased genetic approach; TOP1 counteracts clastogenic activity of G4 ligands by limiting co-transcriptional G4 formation.","method":"Unbiased genetic screen; TOP2 inhibitor treatments; transcription inhibitor experiments; DSB detection assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — unbiased genetic approach plus pharmacological validation with multiple orthogonal drug treatments establishing pathway position","pmids":["34180392"],"is_preprint":false},{"year":2017,"finding":"TOP2A DNA cleavage in human cells is enriched at highly transcribed loci and genes involved in TOP2 poison-related leukemic translocations; TOP2A cleavage cluster regions (CCRs) occur in introns and lincRNA loci and are biased toward distal gene bodies; TOP2 poisons cause a proximal shift in CCR distribution; cleavage correlates independently with both gene length and transcript abundance.","method":"High-throughput sequencing of TOP2A cleavage sites at single-base precision in K562 cells; comparison with ENCODE data for transcription and open chromatin marks; etoposide and other TOP2 poison treatments","journal":"Genome research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — single-base resolution genome-wide mapping with multiple bioinformatic validations against independent ENCODE datasets","pmids":["28385713"],"is_preprint":false},{"year":2016,"finding":"Sister chromatid intertwines (SCIs/catenanes) are formed independently of DNA replication during G2/M by Top2-dependent concatenation of cohesed chromatids due to physical proximity; condensin provides a bias in Top2 function toward decatenation at anaphase onset, as SCI removal in anaphase requires condensin and coincides with hyperactivation of condensin DNA supercoiling activity.","method":"Yeast genetic analysis; 2D gel electrophoresis for SCI detection; condensin and Top2 conditional mutants; cell cycle staging","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with direct biochemical detection of SCIs across multiple cell cycle stages, mechanistically informative","pmids":["27716481"],"is_preprint":false},{"year":2015,"finding":"Condensin relocalization from centromeres to chromosome arms during anaphase requires Polo kinase activity and is followed by Top2 recruitment to chromosome arms in a condensin-dependent manner; this Top2 recruitment coincides with condensin's DNA overwinding activity and promotes chromosome segregation.","method":"ChIP-seq of condensin and Top2 through the cell cycle; conditional Polo kinase inhibition; yeast cell biology with live imaging","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus genetic perturbation experiments in a single lab establishing condensin-dependent Top2 recruitment","pmids":["26686624"],"is_preprint":false},{"year":2011,"finding":"In fission yeast, Top2 is required continuously throughout mitosis including telophase for mitotic chromosome structure; condensin and Top2 have distinct requirements during mitosis—condensin SMC2 mutants accumulate telomeric DNA in lumps at telophase, whereas Top2 mutants show distinct chromosome segregation defects.","method":"Temperature-shift experiments with top2 temperature-sensitive and nda3 cold-sensitive double mutants; cell cycle staging; genetic epistasis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — elegant temperature-shift epistasis experiments in single organism; distinguishes Top2 from condensin roles but single lab","pmids":["21540296"],"is_preprint":false},{"year":1996,"finding":"Human TOP2α and TOP2β can each functionally substitute for yeast Top2 in chromosome segregation, vegetative growth, meiosis, and suppression of rDNA hyper-recombination, demonstrating that isozyme-specific roles of TOP2α in human cells depend on factors extrinsic to catalytic activity.","method":"Complementation of yeast top2 temperature-sensitive and disruption mutants with human TOP2α and TOP2β expression constructs; spore viability; rDNA recombination assays","journal":"Molecular & general genetics : MGG","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional complementation across multiple yeast phenotypes with both human paralogs in parallel, definitively establishing catalytic equivalence","pmids":["8804406"],"is_preprint":false},{"year":1993,"finding":"The top2-5 yeast mutant carrying clustered amino acid substitutions encodes a TOP2 enzyme with reduced amsacrine-stabilized and etoposide-stabilized cleavage in vitro; the mutations identify a domain of the topoisomerase II protein important for interaction with anti-TOP2 anticancer drugs.","method":"Purification of recombinant top2-5 protein; in vitro DNA cleavage assays with amsacrine and etoposide; sequencing of the top2-5 allele; yeast drug sensitivity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical characterization of purified mutant enzyme combined with drug-domain mapping through mutagenesis","pmids":["8395511"],"is_preprint":false},{"year":1995,"finding":"Ser741 of yeast Top2 (homologous to Ser83 of E. coli GyrA) is near a binding site for both quinolone and etoposide; the Ser741→Trp mutation confers quinolone resistance and etoposide hypersensitivity by forming a more stable ternary etoposide-DNA-enzyme complex that is not readily reversible by heat.","method":"Site-directed mutagenesis; purification of mutant Top2 proteins; in vitro DNA cleavage assays with etoposide and quinolone CP-115,953; drug sensitivity assays in yeast","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified site-directed mutant enzyme plus in vivo drug sensitivity, multiple drug classes tested","pmids":["7657608"],"is_preprint":false},{"year":2008,"finding":"Top2 mutants at Pro473 and Gly737 exhibit hypersensitivity to mAMSA; Pro473→Leu generates elevated Top2-mediated single-strand breaks but not double-strand breaks in vitro, and expression of an allele that can only generate single-strand breaks confers mAMSA hypersensitivity in yeast, demonstrating that Top2-generated single-strand breaks can be a component of cell killing.","method":"Mutagenesis screen; purification of mutant Top2 proteins; in vitro DSB and SSB cleavage assays; allele-specific expression in yeast; drug sensitivity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical characterization of purified mutant enzyme combined with allele-specific in vivo genetic validation","pmids":["18723844"],"is_preprint":false},{"year":2018,"finding":"A C-terminally truncated 90-kDa isoform of TOP2α (TOP2α/90), product of intron-19-retaining mRNA, heterodimerizes with full-length TOP2α/170; forced expression of TOP2α/90 in K562 cells suppresses etoposide-mediated DNA strand breaks and cytotoxicity, while siRNA knockdown of TOP2α/90 in resistant cells enhances etoposide-induced DSBs, establishing a dominant-negative mechanism of chemoresistance through heterodimerization.","method":"Co-immunoprecipitation of endogenous TOP2α/90 and TOP2α/170; forced expression and siRNA knockdown; DNA strand break assays; clonogenic survival assays; qPCR and immunoblotting","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous proteins plus bidirectional gain/loss-of-function with mechanistic readout (DNA strand breaks), multiple orthogonal methods in a single study","pmids":["29514855"],"is_preprint":false},{"year":2020,"finding":"Anthracyclines (doxorubicin, epirubicin) and mitoxantrone act as TOP2 poisons at low concentrations but attenuate TOP2-DNA covalent complex formation at higher concentrations, effectively becoming TOP2 inhibitors; TOP2B is the only TOP2 isoform present in iPSC-derived human cardiomyocytes, and doxorubicin does not detectably induce TOP2-DNA complexes in these cells, suggesting inhibition (not poisoning) of TOP2B may underlie cardiotoxicity.","method":"TOP2-DNA complex immunoassays in cells; in vitro DNA cleavage assays with doxorubicin, epirubicin, mitoxantrone, etoposide; iPSC-derived cardiomyocyte immunofluorescence; isoform-specific antibodies","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro cleavage assays plus cell-based complex measurement with isoform-specific detection in physiologically relevant iPSC-derived cardiomyocytes","pmids":["31399497"],"is_preprint":false},{"year":2017,"finding":"Fission yeast Rrp2, an Snf2-family SUMO-targeted DNA translocase, prevents excessive SUMOylation-dependent ubiquitination and proteasomal degradation of Top2; loss of Rrp2 increases Top2 degradation and exposes concealed DNA breaks at Top2-DNA complex sites; Rrp2 competes with the STUbL for SUMO chain binding and displaces SUMOylated Top2 from DNA. The budding yeast homolog Uls1 plays a similar role.","method":"Genome-wide CRISPR/deletion screen; Top2 SUMOylation and ubiquitination assays; Top2 protein stability measurements; DNA damage assays; biochemical SUMO-binding studies","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen plus biochemical SUMO-binding and ubiquitination assays replicated in two yeast species","pmids":["28552615"],"is_preprint":false},{"year":2021,"finding":"VCP/p97 AAA ATPase is required for proteasomal degradation of etoposide-induced TOP2A- and TOP2B-DNA covalent complexes; VCP/p97 inhibition leads to prolonged accumulation of TOP2-DNA complexes in a manner epistatic with the proteasomal pathway and reduces etoposide-induced γH2AX phosphorylation, indicating fewer DSBs are exposed.","method":"TARDIS assay for TOP2-DNA complex quantification; VCP/p97 pharmacological inhibition; epistasis with proteasome inhibitors; γH2AX immunofluorescence","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional epistasis assay with validated TOP2-DNA complex readout, single lab but multiple inhibitor combinations","pmids":["33941661"],"is_preprint":false},{"year":2020,"finding":"Ubiquitin-activating enzyme inhibitors reduce processing of etoposide-induced TOP2A- and TOP2B-DNA covalent complexes; TOP2-DNA complexes are directly conjugated to ubiquitin; inhibition of the Bmi1/Ring1A ubiquitin ligase synergistically enhances TOP2 poison efficacy, establishing ubiquitination as a required step for liberation of protein-free DSBs from TOP2-DNA adducts.","method":"TARDIS assay; ubiquitin-activating enzyme inhibitors; ubiquitin immunoprecipitation of TOP2-DNA complexes; clonogenic survival assays","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct detection of ubiquitinated TOP2-DNA complexes plus functional epistasis, single lab","pmids":["32587095"],"is_preprint":false},{"year":2023,"finding":"Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots at chromatin bridges during cytokinesis; proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated DSB ends; Rad17 then recruits MRN complex and activates ATM-Chk2-INCENP signaling to delay abscission and prevent chromosome breakage, defining the mechanism of the abscission checkpoint.","method":"Live-cell imaging; immunofluorescence for Top2α, Top2ccs, Rad17, MRN, ATM-Chk2-INCENP; proteasome inhibitor treatments; Top2α catalytic mutant expression; siRNA knockdowns","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (live imaging, immunofluorescence, genetic and pharmacological perturbations) establishing mechanistic pathway in a single rigorous study","pmids":["37638884"],"is_preprint":false},{"year":2023,"finding":"RAD54L2 promotes TOP2 cleavage complex (TOP2cc) resolution through a novel mechanism: RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure; this mechanism acts together with ZATT/ZNF451 and independently of TDP2.","method":"Co-immunoprecipitation; ATPase activity assays; genetic epistasis with TDP2 and ZATT knockouts; TOP2cc resolution assays; DNA damage readouts","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical and genetic dissection of a novel pathway with multiple knockouts and co-IP, single study","pmids":["38055822"],"is_preprint":false},{"year":2022,"finding":"TOP2A deficiency in trophoblast cells inhibits proliferation, migration, and invasion and activates the FOXO signaling pathway; TOP2A inhibition in mouse pre-implantation embryos impairs trophectoderm differentiation, embryonic mitochondrial function, and developmental rate; TOP2A expression is lower in villi tissues of RSA patients compared to normal pregnancies.","method":"shRNA knockdown and overexpression in trophoblast cell lines; in vitro proliferation, migration, invasion assays; FOXO pathway western blotting; mouse embryo culture with TOP2A inhibitor; immunofluorescence; immunohistochemistry on clinical samples","journal":"Molecular medicine (Cambridge, Mass.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional genetic manipulation with pathway identification in vitro plus in vivo embryo model, single lab","pmids":["36585615"],"is_preprint":false},{"year":2020,"finding":"Inhibition of TOP2α (using ICRF193 or etoposide) in T cells promotes Top2cc accumulation associated with protein-DNA breaks at genomic DNA, leading to DNA topological disruption and T cell apoptosis; this is linked to diminished TDP2 expression; T cells from patients with chronic viral infection (HBV, HCV, HIV) show lower TOP2α levels and enzymatic activity with Top2cc accumulation.","method":"TOP2α enzymatic activity assays; Top2cc measurement in genomic DNA; TDP2 western blotting; apoptosis assays; Top2α inhibitor treatment of primary T cells","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzymatic activity measurement plus Top2cc detection with functional apoptosis readout, multiple patient cohorts","pmids":["32193368"],"is_preprint":false},{"year":2022,"finding":"In zebrafish, TOP2A mutation causes downregulation of autism-associated genes enriched for PRC2 binding sites and H3K27me3; inhibition of the PRC2 component EZH2 rescues social deficits caused by TOP2A inhibition, placing TOP2A upstream of PRC2/H3K27me3 in an evolutionarily conserved pathway governing social behavior development.","method":"Drug screen in zebrafish; Top2a mutant zebrafish; RNA-seq; chromatin enrichment for H3K27me3 (H3K27me3 ChIP); EZH2 inhibition rescue; behavioral assays in zebrafish and mice","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mutant plus pharmacological rescue with transcriptomic and chromatin data across two model organisms","pmids":["36417527"],"is_preprint":false},{"year":2024,"finding":"CX-5461 (Pol I transcription inhibitor) induces TOP2α-dependent DNA damage preferentially at ribosomal DNA (rDNA) promoter regions, distinct from canonical TOP2α poisons; sensitivity to CX-5461 in murine Eµ-Myc B lymphoma cells is dependent on cellular TOP2α expression/activity.","method":"TOP2α inhibitor and genetic knockdown experiments; RADAR assay for Top2cc detection; γH2AX immunofluorescence at rDNA loci; murine B lymphoma cell line assays; Eµ-Myc model","journal":"Biomedicines","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TOP2cc assay with genomic locus specificity plus genetic dependency in cell line model, single study","pmids":["39062087"],"is_preprint":false},{"year":2022,"finding":"TOP2A deficit in decidualizing endometrial stromal cells leads to abnormal decidualization by activating the NF-κB signaling pathway; TOP2A expression is significantly lower in mid-secretory endometrium of women with recurrent implantation failure; TOP2A-deficient mice showed lower fetal weights.","method":"shRNA knockdown in T-HESCs; mRNA sequencing; NF-κB pathway western blotting; immunofluorescence; in vivo adenovirus-mediated TOP2A knockdown in mice; clinical sample immunohistochemistry","journal":"Reproductive biology and endocrinology : RB&E","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KD with pathway identification by transcriptome sequencing plus in vivo mouse model, single lab","pmids":["36138481"],"is_preprint":false},{"year":2018,"finding":"TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro; depletion of TDP2 in cells does not slow removal of TOP2-DNA complexes, indicating that prior proteolytic processing steps are required before TDP2 acts on remaining 5'-tyrosine adducts. SUMOylation of TOP2 by ZATT E3 ligase is a proteasome-independent mechanism for TOP2cc processing.","method":"TARDIS assay; TDP2 knockdown; in vitro TOP2-DNA complex processing assays; proteasome inhibition; SUMOylated TOP2-DNA complex measurement","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro and cellular assays with negative result for TDP2 on intact TOP2ccs, mechanistically informative negative finding","pmids":["30011940"],"is_preprint":false},{"year":2022,"finding":"GINS1 physically interacts with TOP2A; GINS1 promotes TOP2A protein stability through USP15-mediated deubiquitination, thereby driving glioma cell proliferation and migration; USP15 knockdown reduces TOP2A protein levels and mimics TOP2A loss-of-function.","method":"Co-immunoprecipitation; USP15 knockdown; TOP2A ubiquitination assays; cell proliferation and invasion assays; in vivo xenograft","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus deubiquitinase functional link established, but single lab with limited mechanistic depth","pmids":["36065190"],"is_preprint":false},{"year":2024,"finding":"KDM5B demethylase inhibits ZBTB16 transcription by directly reducing H3K4me3 at the ZBTB16 promoter; reduced ZBTB16 subsequently increases TOP2A expression to confer cisplatin resistance; the deubiquitinase USP7 stabilizes KDM5B by deubiquitination, maintaining this KDM5B/ZBTB16/TOP2A axis in NPC.","method":"ChIP for H3K4me3 at ZBTB16 promoter; KDM5B inhibition/knockdown; ZBTB16 overexpression rescue experiments; USP7 knockdown and ubiquitination assays; cisplatin sensitivity assays in vitro and in vivo","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP establishing direct epigenetic regulation plus multi-step pathway validated by rescue experiments, single lab","pmids":["38287116"],"is_preprint":false},{"year":2015,"finding":"Top2 and Sgs1-Top3 act redundantly to ensure replication fork merging and termination at rDNA barriers; absence of both causes checkpoint activation dependent on the fork barrier protein Fob1 and accumulation of asymmetric X-structures at rDNA; either Top2 or Sgs1-Top3 alone is sufficient to prevent a checkpoint-activating structure at the strongest rDNA barrier.","method":"Yeast genetics; 2D gel electrophoresis for replication intermediates; checkpoint activation assays; Fob1 epistasis; sgs1, top2, top3 deletion combinations","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — systematic yeast genetic epistasis with direct replication intermediate detection, mechanistically defines redundancy between Top2 and RecQ helicase pathways","pmids":["26630413"],"is_preprint":false},{"year":2023,"finding":"EZH2 promotes TOP2A expression by H3K27me3-mediated epigenetic silencing of miR-139-5p; TOP2A is a direct target of miR-139-5p (validated by dual-luciferase reporter assay and ChIP for H3K27me3 at the miR-139-5p locus); inhibition of this axis induces cellular senescence and inhibits HCC cell proliferation in vitro and in vivo.","method":"Dual-luciferase reporter assay; ChIP for H3K27me3 at miR-139-5p promoter; miRNA inhibitor/mimic experiments; siRNA/shRNA knockdown; SA-β-galactosidase senescence assay; xenograft tumor model","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus reporter assay establishing direct epigenetic-miRNA-TOP2A axis, functional validation in vivo, single lab","pmids":["38008711"],"is_preprint":false},{"year":2022,"finding":"E2F1 binds directly to the TOP2A promoter and transcriptionally upregulates TOP2A; E2F1 binding sites in the TOP2A promoter region were confirmed by dual-luciferase reporter and ChIP assays; E2F1 silencing decreases TOP2A levels and inhibits GC cell viability and invasion, effects reversed by TOP2A overexpression.","method":"ChIP assay; dual-luciferase reporter assay; siRNA knockdown of E2F1; TOP2A overexpression rescue; cell viability and invasion assays","journal":"Journal of biosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ChIP plus reporter assay for transcription factor binding, rescue experiments, single lab","pmids":["36550695"],"is_preprint":false},{"year":2015,"finding":"In yeast, TDP1 (tyrosyl-DNA phosphodiesterase 1) participates in repair of Top2α-induced DNA lesions: TDP1-depleted cells accumulate increased amounts of Top2α cleavage complexes after etoposide treatment, show altered kinetics of complex removal, and exhibit increased chromosomal breaks and exchanges; TDP1 acts in NHEJ and alternative end joining but not in homologous recombination for Top2 damage repair.","method":"TDP1 shRNA knockdown; Top2α cleavage complex immunodetection in chromatin; γH2AX and pChk1 assays; cytogenetic analysis; micronucleus assay; DNA-PKcs inhibitor epistasis","journal":"Mutation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TOP2cc chromatin detection plus cytogenetic phenotyping with pathway epistasis, single lab","pmids":["26421495"],"is_preprint":false}],"current_model":"TOP2A encodes a type IIA DNA topoisomerase that resolves DNA topological stress (torsional strain and DNA catenation) at replication forks, transcription units, and during chromosome condensation/segregation by forming transient enzyme-bridged double-strand DNA breaks; its activity and protein stability are regulated by SUMOylation (via ZATT), ubiquitination (via Bmi1/Ring1A, with protection by Rrp2/USP15 and PTEN/OTUD3), proteasomal degradation requiring VCP/p97, and translational control (HuR vs. miR-548c-3p competition at the 3'-UTR); drug-trapped TOP2A-DNA covalent complexes require proteolytic processing before TDP2 can remove residual 5'-phosphotyrosyl adducts, and condensin/ZATT-driven PICH recruitment actively promotes fork reversal and chromosome decatenation in a pathway essential for genome integrity."},"narrative":{"mechanistic_narrative":"TOP2A encodes a type II DNA topoisomerase that resolves DNA topological stress by forming transient enzyme-bridged double-strand breaks, and acts at replication forks, transcription units, and during chromosome condensation and segregation to safeguard genome integrity [PMID:17671091, PMID:27716481]. At replication forks Top2 relieves torsional stress within a narrow window spanning the moving fork and acts coordinately with Top1, with double mutants showing fork block, checkpoint activation, and chromosome breakage [PMID:17671091]; Top2 and the Sgs1-Top3 helicase pathway act redundantly to ensure fork merging and termination at rDNA barriers [PMID:26630413]. Beyond swivel activity, TOP2 changes DNA catenation state to remodel chromatin, synergizing with the BAF (mSWI/SNF) complex to resolve facultative heterochromatin into accessible chromatin [PMID:28250416]. During mitosis Top2 generates and later removes sister chromatid intertwines, with condensin biasing Top2 toward decatenation at anaphase and recruiting Top2 to chromosome arms following Polo kinase–driven condensin relocalization [PMID:27716481, PMID:26686624, PMID:21540296]. The human enzyme is catalytically equivalent to TOP2B, so its isozyme-specific functions depend on extrinsic factors rather than intrinsic catalysis [PMID:8804406]. TOP2A cleavage in human cells is enriched at CTCF-proximal and highly transcribed loci, scaling with gene length and transcript abundance, and these sites coincide with regions involved in TOP2-poison–related leukemic translocations [PMID:31649282, PMID:28385713]. Therapeutic TOP2 poisons (etoposide, amsacrine, anthracyclines) trap covalent TOP2A-DNA cleavage complexes; resolution of these adducts requires proteolytic processing — proteasomal degradation gated by VCP/p97 and by ubiquitination via Bmi1/Ring1A — before TDP2 can remove the residual 5'-phosphotyrosyl adduct, and SUMOylation-dependent pathways (ZATT/ZNF451 with RAD54L2) provide a parallel proteasome-independent route [PMID:24808172, PMID:19956605, PMID:33941661, PMID:32587095, PMID:38055822, PMID:30011940]. TOP2A protein abundance is set by competing ubiquitination and deubiquitination (PTEN/OTUD3, USP15, Rrp2) and by 3'-UTR translational control (HuR versus miR-548c-3p), linking enzyme levels to the G2 decatenation checkpoint, ultra-fine anaphase bridge formation, and an ATM-Chk2 abscission checkpoint during cytokinesis [PMID:33296677, PMID:21768308, PMID:26657567, PMID:28552615, PMID:37638884, PMID:36065190]. TOP2A is also required for trophoblast and endometrial decidualization programs and for an EZH2/PRC2-H3K27me3 axis governing neurodevelopment [PMID:36585615, PMID:36417527, PMID:36138481].","teleology":[{"year":1993,"claim":"Identifying the protein region that mediates drug sensitivity established that TOP2 is the physical target of anticancer poisons and localized the drug-interaction surface.","evidence":"purification and in vitro cleavage assays of the yeast top2-5 mutant enzyme with amsacrine and etoposide","pmids":["8395511"],"confidence":"High","gaps":["Did not resolve atomic structure of the drug-binding pocket","Yeast enzyme, not human TOP2A"]},{"year":1995,"claim":"Mapping Ser741 as a shared quinolone/etoposide contact showed how single residues control ternary cleavage-complex stability and drug reversibility.","evidence":"site-directed mutagenesis and in vitro cleavage assays with etoposide and quinolone CP-115,953","pmids":["7657608"],"confidence":"High","gaps":["Mechanism of complex irreversibility at the structural level unresolved"]},{"year":1996,"claim":"Cross-species complementation answered whether human TOP2A's specialized roles arise from catalysis itself, showing both human isoforms are catalytically interchangeable and isozyme specificity is extrinsic.","evidence":"complementation of yeast top2 mutants with human TOP2A and TOP2B constructs across multiple phenotypes","pmids":["8804406"],"confidence":"High","gaps":["Did not identify the extrinsic factors conferring isozyme specificity"]},{"year":2006,"claim":"Demonstrating Tdp1 acts on 5'-phosphotyrosyl Top2 adducts opened the question of how covalent TOP2-DNA lesions are repaired.","evidence":"yeast deletion analysis plus in vitro biochemistry on Top2 peptide-DNA substrates and drug-sensitivity epistasis","pmids":["16751265"],"confidence":"High","gaps":["Did not establish requirement for prior proteolytic processing","Relative contribution versus other phosphodiesterases unclear"]},{"year":2007,"claim":"Genomic mapping at replication forks defined where and when Top2 relieves torsional stress, establishing its coordinated action with Top1 to prevent fork-associated breakage.","evidence":"ChIP-on-chip of Top1/Top2 on replicating yeast chromosomes with top1/top2 epistasis and Rad53 checkpoint assays","pmids":["17671091"],"confidence":"High","gaps":["Mechanism of sister chromatid junction accumulation not fully resolved","Yeast system"]},{"year":2009,"claim":"Linking Top2 occupancy to transcription-associated fragile loci and identifying Bmi1/Ring1A as the E3 ligase for drug-poisoned TOP2A connected enzyme localization and degradation to genome stability and drug efficacy.","evidence":"ChIP-chip of Top2/Hmo1 with γH2A mapping; siRNA screen plus in vitro ubiquitination and small-molecule ligase inhibition","pmids":["19737516","19956605"],"confidence":"High","gaps":["Whether ubiquitination directly precedes TDP2 action not yet tested","Bmi1 substrate specificity for TOP2A versus other targets"]},{"year":2011,"claim":"Defining the HuR/miR-548c-3p competition at the 3'-UTR and the continuous mitotic requirement for Top2 established translational control of TOP2A abundance and its persistent role through telophase.","evidence":"RNA-IP, MS2-tagged RNA precipitation and reporter assays; yeast temperature-shift epistasis distinguishing Top2 from condensin","pmids":["21768308","21540296"],"confidence":"High","gaps":["Medium confidence for the mitotic-timing finding","How translational control couples to cell-cycle demand unresolved"]},{"year":2014,"claim":"Showing irreversible Top2cc must be proteolytically processed before TDP2 can act, plus a TDP2 crystal structure, defined the ordered processing of trapped complexes and TDP2 substrate accommodation.","evidence":"in vitro Top2cc processing with suicide substrates, TDP2 enzymatic assays, and a 1.6 Å crystal structure","pmids":["24808172"],"confidence":"High","gaps":["Identity of the in vivo protease(s) not defined here","Coupling to cellular degradation machinery unresolved"]},{"year":2015,"claim":"Identifying TOP2-BAF synergy, PTEN/OTUD3-mediated stabilization, Top2/Sgs1-Top3 redundancy, and TDP1 involvement broadened TOP2A function from torsional relief to catenation-based chromatin remodeling, checkpoint control, and break repair.","evidence":"genome-wide ATAC-seq with inhibitors; PTEN/TOP2A co-IP and decatenation checkpoint assays; yeast 2D-gel epistasis; TDP1 knockdown with cytogenetics","pmids":["28250416","26657567","26630413","26421495"],"confidence":"High","gaps":["PTEN/OTUD3 and TDP1 findings are Medium confidence and lack in vitro reconstitution","Mechanism by which catenation state distinguishes heterochromatin from open chromatin unresolved"]},{"year":2016,"claim":"Demonstrating replication-independent formation and condensin-biased anaphase removal of sister chromatid intertwines resolved how Top2 toggles between concatenation and decatenation during the cell cycle.","evidence":"yeast genetics, 2D gels for SCIs, and condensin/Top2 conditional mutants across cell-cycle stages","pmids":["27716481"],"confidence":"High","gaps":["Molecular signal that biases Top2 toward decatenation not fully defined","Yeast system"]},{"year":2017,"claim":"Single-base cleavage mapping, G4-ligand and CX-5461 cytotoxicity dependence, and Rrp2-mediated protection against SUMO-dependent degradation linked TOP2A genomic targeting and stability to drug responses and concealed-break exposure.","evidence":"genome-wide cleavage sequencing in K562; unbiased genetic screens with G4 ligands; CRISPR/deletion screen with SUMO-binding biochemistry in fission yeast","pmids":["28385713","34180392","28552615"],"confidence":"High","gaps":["Determinants of CTCF-proximal versus transcription-proximal cleavage partitioning unresolved","Human ortholog of Rrp2 function not established here"]},{"year":2018,"claim":"Showing TDP2 cannot act on intact Top2ccs and identifying a dominant-negative TOP2A/90 isoform clarified that processing precedes TDP2 and that splice isoforms drive chemoresistance.","evidence":"TARDIS assays with TDP2 knockdown; co-IP and bidirectional gain/loss of function for TOP2α/90","pmids":["30011940","29514855"],"confidence":"High","gaps":["TDP2-processing finding is Medium confidence","Physiological abundance and regulation of TOP2α/90 isoform unclear"]},{"year":2019,"claim":"Defining MDM4 binding and CTCF/transcription-proximal cleavage distribution connected TOP2A to p53 regulation and refined its genomic break landscape relative to chromatin features.","evidence":"co-IP with domain mapping and p53 activity assays; strand-specific nucleotide-resolution cleavage mapping in yeast and human","pmids":["30672125","31649282"],"confidence":"High","gaps":["MDM4 finding is Medium confidence with no in vitro reconstitution","Functional consequence of SSB-prone sites in vivo not fully defined"]},{"year":2020,"claim":"Establishing the ZATT-TOP2A-PICH fork-reversal axis, ubiquitin-required adduct liberation, anthracycline poison-to-inhibitor switching, and TOP2α-dependent T cell apoptosis integrated TOP2A SUMOylation, ubiquitination, and drug pharmacology into genome-protective and pathological pathways.","evidence":"co-IP, PLA, fork-reversal EM and SUMOylation assays; TARDIS with ubiquitin-activating enzyme inhibitors; in vitro cleavage with isoform-specific iPSC-cardiomyocyte assays; enzymatic activity and apoptosis assays in primary T cells","pmids":["33296677","32587095","31399497","32193368"],"confidence":"High","gaps":["Ubiquitination and T-cell findings are Medium confidence","How SUMOylation status routes between PICH recruitment and degradation unresolved"]},{"year":2021,"claim":"Identifying VCP/p97 as required for proteasomal degradation of trapped complexes placed an AAA-ATPase upstream of break exposure in adduct resolution.","evidence":"TARDIS assays with VCP/p97 inhibition, proteasome epistasis, and γH2AX readout","pmids":["33941661"],"confidence":"Medium","gaps":["Single-lab study without reconstitution","How VCP/p97 recognizes TOP2-DNA adducts not defined"]},{"year":2022,"claim":"Establishing TOP2A requirements in trophoblast, decidualization, neurodevelopment, and its E2F1/EZH2/USP15 regulatory wiring extended TOP2A function to developmental programs and tissue-specific transcriptional control.","evidence":"shRNA knockdown with pathway profiling and embryo/mouse models; ChIP and reporter assays for E2F1; zebrafish/mouse behavior with H3K27me3 ChIP; co-IP with USP15-dependent stability assays","pmids":["36585615","36138481","36417527","36550695","36065190"],"confidence":"Medium","gaps":["All findings Medium confidence in single labs","Whether developmental phenotypes reflect catalytic topoisomerase activity or non-catalytic roles is unresolved"]},{"year":2023,"claim":"Defining a cytokinetic abscission checkpoint driven by Top2cc degradation, RAD54L2-mediated SUMO-dependent resolution, and an EZH2/miR-139-5p axis connected TOP2A adduct handling to mitotic checkpoint signaling and tumor proliferation.","evidence":"live imaging and IF for the Rad17-MRN-ATM-Chk2-INCENP pathway; co-IP/ATPase and TDP2/ZATT epistasis for RAD54L2; ChIP, reporter, and senescence/xenograft assays for EZH2/miR-139-5p","pmids":["37638884","38055822","38008711"],"confidence":"Medium","gaps":["RAD54L2 and EZH2 findings are Medium confidence","Relationship between TDP2-independent and TDP2-dependent resolution pathways in vivo not resolved"]},{"year":2024,"claim":"Identifying TOP2α-dependent rDNA-promoter damage from CX-5461 and a KDM5B/ZBTB16 axis controlling TOP2A in chemoresistance extended TOP2A targeting and regulation to nucleolar damage and epigenetic drug-resistance circuits.","evidence":"RADAR/Top2cc assays with genetic dependency in Eµ-Myc lymphoma; ChIP for H3K4me3 with USP7 ubiquitination and cisplatin sensitivity assays","pmids":["39062087","38287116"],"confidence":"Medium","gaps":["Both findings Medium confidence in single labs","Generality of rDNA-localized TOP2α damage across cell types unknown"]},{"year":null,"claim":"It remains unresolved which proteases and how the choice between proteasomal versus SUMO-dependent (ZATT/RAD54L2) resolution routes is made for trapped TOP2A-DNA complexes, and what extrinsic factors confer TOP2A isozyme specificity in human cells.","evidence":"","pmids":[],"confidence":"Medium","gaps":["In vivo protease(s) for Top2cc processing unidentified","Determinants routing SUMOylated TOP2A to PICH recruitment versus degradation undefined","Extrinsic isozyme-specificity factors not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0,6,13,19]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,6,17]},{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[5,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,3,6,25]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[13,14,15]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[30]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[9,13,25]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0,7,35]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,2,23,26]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[5,29]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[12,20,33]}],"complexes":[],"partners":["PICH","ZATT","PTEN","OTUD3","MDM4","GINS1","RAD54L2","TDP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P11388","full_name":"DNA topoisomerase 2-alpha","aliases":["DNA topoisomerase II, alpha isozyme"],"length_aa":1531,"mass_kda":174.4,"function":"Key decatenating enzyme that alters DNA topology by binding to two double-stranded DNA molecules, generating a double-stranded break in one of the strands, passing the intact strand through the broken strand, and religating the broken strand (PubMed:17567603, PubMed:18790802, PubMed:22013166, PubMed:22323612, PubMed:27754753). 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MCPH30","url":"https://www.omim.org/entry/620183"},{"mim_id":"615385","title":"MICRO RNA 485; MIR485","url":"https://www.omim.org/entry/615385"},{"mim_id":"609683","title":"DNA CROSS-LINK REPAIR PROTEIN 1B; DCLRE1B","url":"https://www.omim.org/entry/609683"},{"mim_id":"609296","title":"B-CELL IMMUNODEFICIENCY, DISTAL LIMB ANOMALIES, AND UROGENITAL MALFORMATIONS; BILU","url":"https://www.omim.org/entry/609296"},{"mim_id":"609100","title":"F-BOX ONLY PROTEIN 28; FBXO28","url":"https://www.omim.org/entry/609100"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":119.0},{"tissue":"testis","ntpm":62.4}],"url":"https://www.proteinatlas.org/search/TOP2A"},"hgnc":{"alias_symbol":["TOP2alpha","TOPIIA"],"prev_symbol":["TOP2"]},"alphafold":{"accession":"P11388","domains":[{"cath_id":"3.30.565.10","chopping":"34-265","consensus_level":"medium","plddt":91.5404,"start":34,"end":265},{"cath_id":"3.30.230.10","chopping":"281-423","consensus_level":"medium","plddt":88.9571,"start":281,"end":423},{"cath_id":"3.40.50.670","chopping":"427-576_616-703","consensus_level":"high","plddt":88.9311,"start":427,"end":703},{"cath_id":"3.30.1490","chopping":"577-613","consensus_level":"medium","plddt":84.1111,"start":577,"end":613},{"cath_id":"3.30.1360.40","chopping":"899-997","consensus_level":"medium","plddt":91.2708,"start":899,"end":997}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P11388","model_url":"https://alphafold.ebi.ac.uk/files/AF-P11388-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P11388-F1-predicted_aligned_error_v6.png","plddt_mean":74.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TOP2A","jax_strain_url":"https://www.jax.org/strain/search?query=TOP2A"},"sequence":{"accession":"P11388","fasta_url":"https://rest.uniprot.org/uniprotkb/P11388.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P11388/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P11388"}},"corpus_meta":[{"pmid":"36322662","id":"PMC_36322662","title":"Untangling the roles of TOP2A and TOP2B in transcription and cancer.","date":"2022","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/36322662","citation_count":176,"is_preprint":false},{"pmid":"17671091","id":"PMC_17671091","title":"Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation.","date":"2007","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/17671091","citation_count":136,"is_preprint":false},{"pmid":"16751265","id":"PMC_16751265","title":"Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage.","date":"2006","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/16751265","citation_count":129,"is_preprint":false},{"pmid":"18332872","id":"PMC_18332872","title":"Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines.","date":"2008","source":"Laboratory investigation; 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Top2 at replication forks.\",\n      \"method\": \"ChIP-on-chip mapping of Top1/Top2 on replicating yeast chromosomes; genetic analysis of top1, top2, and top1 top2 double mutants; checkpoint kinase (Rad53) phosphorylation assays; Exo1 epistasis experiments\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genomic mapping plus multi-gene epistasis, replicated across multiple mutant combinations in a single rigorous study\",\n      \"pmids\": [\"17671091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Tyrosyl-DNA phosphodiesterase (Tdp1) participates in repair of Top2-mediated DNA damage: Tdp1 can remove a Top2-derived peptide covalently linked to DNA via a 5'-phosphotyrosyl bond in vitro; tdp1 deletion confers hypersensitivity to Top2-targeting drugs; Tdp1 acts in collaboration with NHEJ, excision repair, and post-replication repair pathways.\",\n      \"method\": \"Yeast genetic deletion analysis; in vitro biochemical assay with bacterially expressed Tdp1p acting on 5'-phosphotyrosyl-linked Top2 peptide-DNA substrate; drug sensitivity assays; double-mutant epistasis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of enzymatic activity plus genetic epistasis across multiple pathways\",\n      \"pmids\": [\"16751265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Irreversible Top2-DNA covalent complexes (Top2cc) require proteolytic processing (proteasomal degradation or denaturation) before TDP2 can remove the remaining 5'-phosphotyrosyl adduct; TDP2 is most active when the tyrosyl-linked DNA is single-stranded; TDP2 can also process tyrosine linked to RNA substrates. A 1.6 Å crystal structure of TDP2 bound to a 5'-ribonucleotide-bearing substrate explains RNA accommodation in the active site.\",\n      \"method\": \"In vitro TOP2cc processing assays with suicidal substrates; TDP2 enzymatic assays with varied substrates; crystal structure at 1.6 Å resolution; protease/denaturation pre-treatment experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mechanistic substrate variants plus high-resolution crystal structure in a single study\",\n      \"pmids\": [\"24808172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Top2 binds intergenic regions near transcribed genes specifically in S phase; Top2-bound loci exhibit low nucleosome density; loss of Top2 causes γH2A accumulation at these loci; HMG protein Hmo1 co-occupies these loci and is deleterious in top2 mutants. Top2 is dispensable for transcription per se but suppresses chromosome fragility at M-G1 transition at transcription-associated loci.\",\n      \"method\": \"ChIP-chip mapping of Top2 and Hmo1 across yeast cell cycle; γH2A ChIP; genetic analysis of top2 and hmo1 mutants; nucleosome occupancy assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide ChIP-chip plus genetic epistasis with multiple chromatin markers; published in high-impact journal with multiple orthogonal methods\",\n      \"pmids\": [\"19737516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Drug-poisoned TOP2α undergoes proteasomal degradation; the E3 ubiquitin ligase Bmi1/Ring1A ubiquitinates TOP2α in vitro and in cells; siRNA silencing of Bmi1 inhibits drug-induced TOP2α degradation and increases TOP2α-DNA cleavage complex persistence and drug efficacy. A small-molecule inhibitor of Bmi1/Ring1A ubiquitination activity prevents TOP2α ubiquitination and drug-induced degradation, synergistically enhancing TOP2 poison efficacy.\",\n      \"method\": \"siRNA screen identifying Bmi1/Ring1A; in vitro ubiquitination assay; cellular overexpression of Bmi1; drug sensitivity assays; small-molecule inhibitor of ubiquitin ligase\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro ubiquitination reconstitution plus cellular siRNA and overexpression experiments with functional readouts, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"19956605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TOP2 (but not TOP1) synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin, independent of transcription; this indicates that DNA decatenation/catenation topology changes (not torsional swiveling) are required for heterochromatin resolution. TOP2 also plays a role in re-formation of facultative heterochromatin, suggesting heterochromatin and accessible chromatin differ in catenation states.\",\n      \"method\": \"Genome-wide ATAC-seq and chromatin accessibility assays; TOP2 and TOP1 inhibitor treatments; BAF complex genetic perturbations; in vivo chromatin remodeling assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide chromatin accessibility mapping with multiple pharmacological and genetic perturbations; mechanistic distinction between TOP1 and TOP2 functions established\",\n      \"pmids\": [\"28250416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TOP2A cleavage activity in humans is distributed in two fractions: tightly localized CTCF-proximal sites and broadly distributed transcription-proximal sites (correlated with gene length and transcript abundance); single-nucleotide mapping distinguishes canonical DSB sites from strand-biased SSB-prone sites induced by etoposide; Mre11-dependent repair of Top2 breaks was characterized in yeast.\",\n      \"method\": \"Strand-specific nucleotide-resolution mapping of Top2 DNA cleavage (END-seq derivative) in S. cerevisiae and human genomes; comparison with ENCODE chromatin marks; etoposide treatment; meiotic Spo11 validation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — nucleotide-resolution genome-wide mapping across two organisms with multiple biological validations and functional annotations\",\n      \"pmids\": [\"31649282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Upon replication stress, TOP2A is recruited to stalled replication forks in a manner dependent on HLTF, ZRANB3, and SMARCAL1; TOP2A undergoes SUMOylation mediated by the SUMO E3 ligase ZATT; SUMOylated TOP2A then recruits the SUMO-targeted DNA translocase PICH; this ZATT-TOP2A-PICH axis drives extensive fork reversal by resolving topological barriers. Loss of this axis causes accumulation of partially reversed forks and genome instability.\",\n      \"method\": \"Co-immunoprecipitation; proximity ligation assays; siRNA knockdown of HLTF/ZRANB3/SMARCAL1/ZATT/PICH; fork reversal electron microscopy; SUMOylation assays; genome instability measurements\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple reciprocal co-IPs, SUMOylation assays, genetic knockdowns with electron microscopy phenotypic readout, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"33296677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HuR binds the TOP2A 3'-UTR and increases TOP2A translation; reducing HuR triggers recruitment of TOP2A mRNA to RISC components and cytoplasmic processing bodies; miR-548c-3p, identified by MS2-tagged RNA precipitation, mediates repression of TOP2A translation by antagonizing HuR; lowering TOP2A by HuR silencing or miR-548c-3p overexpression decreases DNA damage after doxorubicin treatment.\",\n      \"method\": \"RNA-binding protein immunoprecipitation; MS2-tagged RNA precipitation to identify miR-548c-3p; reporter assays; siRNA knockdown of HuR; miRNA overexpression; doxorubicin cytotoxicity assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal RNA-protein interaction methods plus functional validation with drug treatment in a single study\",\n      \"pmids\": [\"21768308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PTEN physically associates with TOP2A and stabilizes it through the deubiquitinase OTUD3; in PTEN-deficient cells, TOP2A ubiquitination increases and TOP2A protein levels decrease, leading to defective DNA decatenation checkpoint in G2, accumulation of ultra-fine anaphase bridges, and incomplete DNA decatenation.\",\n      \"method\": \"Co-immunoprecipitation of PTEN and TOP2A; ubiquitination assays; OTUD3 knockdown; analysis of ultra-fine bridges; decatenation checkpoint assays in PTEN-null cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional cellular assays in a single lab, multiple readouts but no in vitro reconstitution\",\n      \"pmids\": [\"26657567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MDM4 and TOP2A physically bind each other; the C-terminal region (CTR) of TOP2A binds residues 188–238 of MDM4; this interaction stabilizes TOP2A protein post-translationally, and TOP2A binding activates MDM4 for p53 binding, resulting in enhanced p53 inhibition and increased cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation; domain-mapping experiments with truncation constructs; siRNA knockdown; cell proliferation assays; p53 activity assays\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP with domain mapping in a single lab, functional validation by KD/OE but no in vitro reconstitution\",\n      \"pmids\": [\"30672125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TOP2 activity and transcription both contribute to DNA double-strand break formation after G4 ligand (pyridostatin and CX-5461) treatment; TOP2A was identified as a major effector of cytotoxicity by an unbiased genetic approach; TOP1 counteracts clastogenic activity of G4 ligands by limiting co-transcriptional G4 formation.\",\n      \"method\": \"Unbiased genetic screen; TOP2 inhibitor treatments; transcription inhibitor experiments; DSB detection assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased genetic approach plus pharmacological validation with multiple orthogonal drug treatments establishing pathway position\",\n      \"pmids\": [\"34180392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TOP2A DNA cleavage in human cells is enriched at highly transcribed loci and genes involved in TOP2 poison-related leukemic translocations; TOP2A cleavage cluster regions (CCRs) occur in introns and lincRNA loci and are biased toward distal gene bodies; TOP2 poisons cause a proximal shift in CCR distribution; cleavage correlates independently with both gene length and transcript abundance.\",\n      \"method\": \"High-throughput sequencing of TOP2A cleavage sites at single-base precision in K562 cells; comparison with ENCODE data for transcription and open chromatin marks; etoposide and other TOP2 poison treatments\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single-base resolution genome-wide mapping with multiple bioinformatic validations against independent ENCODE datasets\",\n      \"pmids\": [\"28385713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Sister chromatid intertwines (SCIs/catenanes) are formed independently of DNA replication during G2/M by Top2-dependent concatenation of cohesed chromatids due to physical proximity; condensin provides a bias in Top2 function toward decatenation at anaphase onset, as SCI removal in anaphase requires condensin and coincides with hyperactivation of condensin DNA supercoiling activity.\",\n      \"method\": \"Yeast genetic analysis; 2D gel electrophoresis for SCI detection; condensin and Top2 conditional mutants; cell cycle staging\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with direct biochemical detection of SCIs across multiple cell cycle stages, mechanistically informative\",\n      \"pmids\": [\"27716481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Condensin relocalization from centromeres to chromosome arms during anaphase requires Polo kinase activity and is followed by Top2 recruitment to chromosome arms in a condensin-dependent manner; this Top2 recruitment coincides with condensin's DNA overwinding activity and promotes chromosome segregation.\",\n      \"method\": \"ChIP-seq of condensin and Top2 through the cell cycle; conditional Polo kinase inhibition; yeast cell biology with live imaging\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus genetic perturbation experiments in a single lab establishing condensin-dependent Top2 recruitment\",\n      \"pmids\": [\"26686624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In fission yeast, Top2 is required continuously throughout mitosis including telophase for mitotic chromosome structure; condensin and Top2 have distinct requirements during mitosis—condensin SMC2 mutants accumulate telomeric DNA in lumps at telophase, whereas Top2 mutants show distinct chromosome segregation defects.\",\n      \"method\": \"Temperature-shift experiments with top2 temperature-sensitive and nda3 cold-sensitive double mutants; cell cycle staging; genetic epistasis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — elegant temperature-shift epistasis experiments in single organism; distinguishes Top2 from condensin roles but single lab\",\n      \"pmids\": [\"21540296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human TOP2α and TOP2β can each functionally substitute for yeast Top2 in chromosome segregation, vegetative growth, meiosis, and suppression of rDNA hyper-recombination, demonstrating that isozyme-specific roles of TOP2α in human cells depend on factors extrinsic to catalytic activity.\",\n      \"method\": \"Complementation of yeast top2 temperature-sensitive and disruption mutants with human TOP2α and TOP2β expression constructs; spore viability; rDNA recombination assays\",\n      \"journal\": \"Molecular & general genetics : MGG\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional complementation across multiple yeast phenotypes with both human paralogs in parallel, definitively establishing catalytic equivalence\",\n      \"pmids\": [\"8804406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"The top2-5 yeast mutant carrying clustered amino acid substitutions encodes a TOP2 enzyme with reduced amsacrine-stabilized and etoposide-stabilized cleavage in vitro; the mutations identify a domain of the topoisomerase II protein important for interaction with anti-TOP2 anticancer drugs.\",\n      \"method\": \"Purification of recombinant top2-5 protein; in vitro DNA cleavage assays with amsacrine and etoposide; sequencing of the top2-5 allele; yeast drug sensitivity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical characterization of purified mutant enzyme combined with drug-domain mapping through mutagenesis\",\n      \"pmids\": [\"8395511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Ser741 of yeast Top2 (homologous to Ser83 of E. coli GyrA) is near a binding site for both quinolone and etoposide; the Ser741→Trp mutation confers quinolone resistance and etoposide hypersensitivity by forming a more stable ternary etoposide-DNA-enzyme complex that is not readily reversible by heat.\",\n      \"method\": \"Site-directed mutagenesis; purification of mutant Top2 proteins; in vitro DNA cleavage assays with etoposide and quinolone CP-115,953; drug sensitivity assays in yeast\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified site-directed mutant enzyme plus in vivo drug sensitivity, multiple drug classes tested\",\n      \"pmids\": [\"7657608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Top2 mutants at Pro473 and Gly737 exhibit hypersensitivity to mAMSA; Pro473→Leu generates elevated Top2-mediated single-strand breaks but not double-strand breaks in vitro, and expression of an allele that can only generate single-strand breaks confers mAMSA hypersensitivity in yeast, demonstrating that Top2-generated single-strand breaks can be a component of cell killing.\",\n      \"method\": \"Mutagenesis screen; purification of mutant Top2 proteins; in vitro DSB and SSB cleavage assays; allele-specific expression in yeast; drug sensitivity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical characterization of purified mutant enzyme combined with allele-specific in vivo genetic validation\",\n      \"pmids\": [\"18723844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A C-terminally truncated 90-kDa isoform of TOP2α (TOP2α/90), product of intron-19-retaining mRNA, heterodimerizes with full-length TOP2α/170; forced expression of TOP2α/90 in K562 cells suppresses etoposide-mediated DNA strand breaks and cytotoxicity, while siRNA knockdown of TOP2α/90 in resistant cells enhances etoposide-induced DSBs, establishing a dominant-negative mechanism of chemoresistance through heterodimerization.\",\n      \"method\": \"Co-immunoprecipitation of endogenous TOP2α/90 and TOP2α/170; forced expression and siRNA knockdown; DNA strand break assays; clonogenic survival assays; qPCR and immunoblotting\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous proteins plus bidirectional gain/loss-of-function with mechanistic readout (DNA strand breaks), multiple orthogonal methods in a single study\",\n      \"pmids\": [\"29514855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Anthracyclines (doxorubicin, epirubicin) and mitoxantrone act as TOP2 poisons at low concentrations but attenuate TOP2-DNA covalent complex formation at higher concentrations, effectively becoming TOP2 inhibitors; TOP2B is the only TOP2 isoform present in iPSC-derived human cardiomyocytes, and doxorubicin does not detectably induce TOP2-DNA complexes in these cells, suggesting inhibition (not poisoning) of TOP2B may underlie cardiotoxicity.\",\n      \"method\": \"TOP2-DNA complex immunoassays in cells; in vitro DNA cleavage assays with doxorubicin, epirubicin, mitoxantrone, etoposide; iPSC-derived cardiomyocyte immunofluorescence; isoform-specific antibodies\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro cleavage assays plus cell-based complex measurement with isoform-specific detection in physiologically relevant iPSC-derived cardiomyocytes\",\n      \"pmids\": [\"31399497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Fission yeast Rrp2, an Snf2-family SUMO-targeted DNA translocase, prevents excessive SUMOylation-dependent ubiquitination and proteasomal degradation of Top2; loss of Rrp2 increases Top2 degradation and exposes concealed DNA breaks at Top2-DNA complex sites; Rrp2 competes with the STUbL for SUMO chain binding and displaces SUMOylated Top2 from DNA. The budding yeast homolog Uls1 plays a similar role.\",\n      \"method\": \"Genome-wide CRISPR/deletion screen; Top2 SUMOylation and ubiquitination assays; Top2 protein stability measurements; DNA damage assays; biochemical SUMO-binding studies\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen plus biochemical SUMO-binding and ubiquitination assays replicated in two yeast species\",\n      \"pmids\": [\"28552615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"VCP/p97 AAA ATPase is required for proteasomal degradation of etoposide-induced TOP2A- and TOP2B-DNA covalent complexes; VCP/p97 inhibition leads to prolonged accumulation of TOP2-DNA complexes in a manner epistatic with the proteasomal pathway and reduces etoposide-induced γH2AX phosphorylation, indicating fewer DSBs are exposed.\",\n      \"method\": \"TARDIS assay for TOP2-DNA complex quantification; VCP/p97 pharmacological inhibition; epistasis with proteasome inhibitors; γH2AX immunofluorescence\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional epistasis assay with validated TOP2-DNA complex readout, single lab but multiple inhibitor combinations\",\n      \"pmids\": [\"33941661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Ubiquitin-activating enzyme inhibitors reduce processing of etoposide-induced TOP2A- and TOP2B-DNA covalent complexes; TOP2-DNA complexes are directly conjugated to ubiquitin; inhibition of the Bmi1/Ring1A ubiquitin ligase synergistically enhances TOP2 poison efficacy, establishing ubiquitination as a required step for liberation of protein-free DSBs from TOP2-DNA adducts.\",\n      \"method\": \"TARDIS assay; ubiquitin-activating enzyme inhibitors; ubiquitin immunoprecipitation of TOP2-DNA complexes; clonogenic survival assays\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct detection of ubiquitinated TOP2-DNA complexes plus functional epistasis, single lab\",\n      \"pmids\": [\"32587095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots at chromatin bridges during cytokinesis; proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated DSB ends; Rad17 then recruits MRN complex and activates ATM-Chk2-INCENP signaling to delay abscission and prevent chromosome breakage, defining the mechanism of the abscission checkpoint.\",\n      \"method\": \"Live-cell imaging; immunofluorescence for Top2α, Top2ccs, Rad17, MRN, ATM-Chk2-INCENP; proteasome inhibitor treatments; Top2α catalytic mutant expression; siRNA knockdowns\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (live imaging, immunofluorescence, genetic and pharmacological perturbations) establishing mechanistic pathway in a single rigorous study\",\n      \"pmids\": [\"37638884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RAD54L2 promotes TOP2 cleavage complex (TOP2cc) resolution through a novel mechanism: RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure; this mechanism acts together with ZATT/ZNF451 and independently of TDP2.\",\n      \"method\": \"Co-immunoprecipitation; ATPase activity assays; genetic epistasis with TDP2 and ZATT knockouts; TOP2cc resolution assays; DNA damage readouts\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical and genetic dissection of a novel pathway with multiple knockouts and co-IP, single study\",\n      \"pmids\": [\"38055822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TOP2A deficiency in trophoblast cells inhibits proliferation, migration, and invasion and activates the FOXO signaling pathway; TOP2A inhibition in mouse pre-implantation embryos impairs trophectoderm differentiation, embryonic mitochondrial function, and developmental rate; TOP2A expression is lower in villi tissues of RSA patients compared to normal pregnancies.\",\n      \"method\": \"shRNA knockdown and overexpression in trophoblast cell lines; in vitro proliferation, migration, invasion assays; FOXO pathway western blotting; mouse embryo culture with TOP2A inhibitor; immunofluorescence; immunohistochemistry on clinical samples\",\n      \"journal\": \"Molecular medicine (Cambridge, Mass.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional genetic manipulation with pathway identification in vitro plus in vivo embryo model, single lab\",\n      \"pmids\": [\"36585615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Inhibition of TOP2α (using ICRF193 or etoposide) in T cells promotes Top2cc accumulation associated with protein-DNA breaks at genomic DNA, leading to DNA topological disruption and T cell apoptosis; this is linked to diminished TDP2 expression; T cells from patients with chronic viral infection (HBV, HCV, HIV) show lower TOP2α levels and enzymatic activity with Top2cc accumulation.\",\n      \"method\": \"TOP2α enzymatic activity assays; Top2cc measurement in genomic DNA; TDP2 western blotting; apoptosis assays; Top2α inhibitor treatment of primary T cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic activity measurement plus Top2cc detection with functional apoptosis readout, multiple patient cohorts\",\n      \"pmids\": [\"32193368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In zebrafish, TOP2A mutation causes downregulation of autism-associated genes enriched for PRC2 binding sites and H3K27me3; inhibition of the PRC2 component EZH2 rescues social deficits caused by TOP2A inhibition, placing TOP2A upstream of PRC2/H3K27me3 in an evolutionarily conserved pathway governing social behavior development.\",\n      \"method\": \"Drug screen in zebrafish; Top2a mutant zebrafish; RNA-seq; chromatin enrichment for H3K27me3 (H3K27me3 ChIP); EZH2 inhibition rescue; behavioral assays in zebrafish and mice\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mutant plus pharmacological rescue with transcriptomic and chromatin data across two model organisms\",\n      \"pmids\": [\"36417527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CX-5461 (Pol I transcription inhibitor) induces TOP2α-dependent DNA damage preferentially at ribosomal DNA (rDNA) promoter regions, distinct from canonical TOP2α poisons; sensitivity to CX-5461 in murine Eµ-Myc B lymphoma cells is dependent on cellular TOP2α expression/activity.\",\n      \"method\": \"TOP2α inhibitor and genetic knockdown experiments; RADAR assay for Top2cc detection; γH2AX immunofluorescence at rDNA loci; murine B lymphoma cell line assays; Eµ-Myc model\",\n      \"journal\": \"Biomedicines\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TOP2cc assay with genomic locus specificity plus genetic dependency in cell line model, single study\",\n      \"pmids\": [\"39062087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TOP2A deficit in decidualizing endometrial stromal cells leads to abnormal decidualization by activating the NF-κB signaling pathway; TOP2A expression is significantly lower in mid-secretory endometrium of women with recurrent implantation failure; TOP2A-deficient mice showed lower fetal weights.\",\n      \"method\": \"shRNA knockdown in T-HESCs; mRNA sequencing; NF-κB pathway western blotting; immunofluorescence; in vivo adenovirus-mediated TOP2A knockdown in mice; clinical sample immunohistochemistry\",\n      \"journal\": \"Reproductive biology and endocrinology : RB&E\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KD with pathway identification by transcriptome sequencing plus in vivo mouse model, single lab\",\n      \"pmids\": [\"36138481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro; depletion of TDP2 in cells does not slow removal of TOP2-DNA complexes, indicating that prior proteolytic processing steps are required before TDP2 acts on remaining 5'-tyrosine adducts. SUMOylation of TOP2 by ZATT E3 ligase is a proteasome-independent mechanism for TOP2cc processing.\",\n      \"method\": \"TARDIS assay; TDP2 knockdown; in vitro TOP2-DNA complex processing assays; proteasome inhibition; SUMOylated TOP2-DNA complex measurement\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro and cellular assays with negative result for TDP2 on intact TOP2ccs, mechanistically informative negative finding\",\n      \"pmids\": [\"30011940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GINS1 physically interacts with TOP2A; GINS1 promotes TOP2A protein stability through USP15-mediated deubiquitination, thereby driving glioma cell proliferation and migration; USP15 knockdown reduces TOP2A protein levels and mimics TOP2A loss-of-function.\",\n      \"method\": \"Co-immunoprecipitation; USP15 knockdown; TOP2A ubiquitination assays; cell proliferation and invasion assays; in vivo xenograft\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus deubiquitinase functional link established, but single lab with limited mechanistic depth\",\n      \"pmids\": [\"36065190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KDM5B demethylase inhibits ZBTB16 transcription by directly reducing H3K4me3 at the ZBTB16 promoter; reduced ZBTB16 subsequently increases TOP2A expression to confer cisplatin resistance; the deubiquitinase USP7 stabilizes KDM5B by deubiquitination, maintaining this KDM5B/ZBTB16/TOP2A axis in NPC.\",\n      \"method\": \"ChIP for H3K4me3 at ZBTB16 promoter; KDM5B inhibition/knockdown; ZBTB16 overexpression rescue experiments; USP7 knockdown and ubiquitination assays; cisplatin sensitivity assays in vitro and in vivo\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP establishing direct epigenetic regulation plus multi-step pathway validated by rescue experiments, single lab\",\n      \"pmids\": [\"38287116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Top2 and Sgs1-Top3 act redundantly to ensure replication fork merging and termination at rDNA barriers; absence of both causes checkpoint activation dependent on the fork barrier protein Fob1 and accumulation of asymmetric X-structures at rDNA; either Top2 or Sgs1-Top3 alone is sufficient to prevent a checkpoint-activating structure at the strongest rDNA barrier.\",\n      \"method\": \"Yeast genetics; 2D gel electrophoresis for replication intermediates; checkpoint activation assays; Fob1 epistasis; sgs1, top2, top3 deletion combinations\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic yeast genetic epistasis with direct replication intermediate detection, mechanistically defines redundancy between Top2 and RecQ helicase pathways\",\n      \"pmids\": [\"26630413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EZH2 promotes TOP2A expression by H3K27me3-mediated epigenetic silencing of miR-139-5p; TOP2A is a direct target of miR-139-5p (validated by dual-luciferase reporter assay and ChIP for H3K27me3 at the miR-139-5p locus); inhibition of this axis induces cellular senescence and inhibits HCC cell proliferation in vitro and in vivo.\",\n      \"method\": \"Dual-luciferase reporter assay; ChIP for H3K27me3 at miR-139-5p promoter; miRNA inhibitor/mimic experiments; siRNA/shRNA knockdown; SA-β-galactosidase senescence assay; xenograft tumor model\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus reporter assay establishing direct epigenetic-miRNA-TOP2A axis, functional validation in vivo, single lab\",\n      \"pmids\": [\"38008711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"E2F1 binds directly to the TOP2A promoter and transcriptionally upregulates TOP2A; E2F1 binding sites in the TOP2A promoter region were confirmed by dual-luciferase reporter and ChIP assays; E2F1 silencing decreases TOP2A levels and inhibits GC cell viability and invasion, effects reversed by TOP2A overexpression.\",\n      \"method\": \"ChIP assay; dual-luciferase reporter assay; siRNA knockdown of E2F1; TOP2A overexpression rescue; cell viability and invasion assays\",\n      \"journal\": \"Journal of biosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ChIP plus reporter assay for transcription factor binding, rescue experiments, single lab\",\n      \"pmids\": [\"36550695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In yeast, TDP1 (tyrosyl-DNA phosphodiesterase 1) participates in repair of Top2α-induced DNA lesions: TDP1-depleted cells accumulate increased amounts of Top2α cleavage complexes after etoposide treatment, show altered kinetics of complex removal, and exhibit increased chromosomal breaks and exchanges; TDP1 acts in NHEJ and alternative end joining but not in homologous recombination for Top2 damage repair.\",\n      \"method\": \"TDP1 shRNA knockdown; Top2α cleavage complex immunodetection in chromatin; γH2AX and pChk1 assays; cytogenetic analysis; micronucleus assay; DNA-PKcs inhibitor epistasis\",\n      \"journal\": \"Mutation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TOP2cc chromatin detection plus cytogenetic phenotyping with pathway epistasis, single lab\",\n      \"pmids\": [\"26421495\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TOP2A encodes a type IIA DNA topoisomerase that resolves DNA topological stress (torsional strain and DNA catenation) at replication forks, transcription units, and during chromosome condensation/segregation by forming transient enzyme-bridged double-strand DNA breaks; its activity and protein stability are regulated by SUMOylation (via ZATT), ubiquitination (via Bmi1/Ring1A, with protection by Rrp2/USP15 and PTEN/OTUD3), proteasomal degradation requiring VCP/p97, and translational control (HuR vs. miR-548c-3p competition at the 3'-UTR); drug-trapped TOP2A-DNA covalent complexes require proteolytic processing before TDP2 can remove residual 5'-phosphotyrosyl adducts, and condensin/ZATT-driven PICH recruitment actively promotes fork reversal and chromosome decatenation in a pathway essential for genome integrity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TOP2A encodes a type II DNA topoisomerase that resolves DNA topological stress by forming transient enzyme-bridged double-strand breaks, and acts at replication forks, transcription units, and during chromosome condensation and segregation to safeguard genome integrity [#0, #13]. At replication forks Top2 relieves torsional stress within a narrow window spanning the moving fork and acts coordinately with Top1, with double mutants showing fork block, checkpoint activation, and chromosome breakage [#0]; Top2 and the Sgs1-Top3 helicase pathway act redundantly to ensure fork merging and termination at rDNA barriers [#35]. Beyond swivel activity, TOP2 changes DNA catenation state to remodel chromatin, synergizing with the BAF (mSWI/SNF) complex to resolve facultative heterochromatin into accessible chromatin [#5]. During mitosis Top2 generates and later removes sister chromatid intertwines, with condensin biasing Top2 toward decatenation at anaphase and recruiting Top2 to chromosome arms following Polo kinase–driven condensin relocalization [#13, #14, #15]. The human enzyme is catalytically equivalent to TOP2B, so its isozyme-specific functions depend on extrinsic factors rather than intrinsic catalysis [#16]. TOP2A cleavage in human cells is enriched at CTCF-proximal and highly transcribed loci, scaling with gene length and transcript abundance, and these sites coincide with regions involved in TOP2-poison–related leukemic translocations [#6, #12]. Therapeutic TOP2 poisons (etoposide, amsacrine, anthracyclines) trap covalent TOP2A-DNA cleavage complexes; resolution of these adducts requires proteolytic processing — proteasomal degradation gated by VCP/p97 and by ubiquitination via Bmi1/Ring1A — before TDP2 can remove the residual 5'-phosphotyrosyl adduct, and SUMOylation-dependent pathways (ZATT/ZNF451 with RAD54L2) provide a parallel proteasome-independent route [#2, #4, #23, #24, #26, #32]. TOP2A protein abundance is set by competing ubiquitination and deubiquitination (PTEN/OTUD3, USP15, Rrp2) and by 3'-UTR translational control (HuR versus miR-548c-3p), linking enzyme levels to the G2 decatenation checkpoint, ultra-fine anaphase bridge formation, and an ATM-Chk2 abscission checkpoint during cytokinesis [#7, #8, #9, #22, #25, #33]. TOP2A is also required for trophoblast and endometrial decidualization programs and for an EZH2/PRC2-H3K27me3 axis governing neurodevelopment [#27, #29, #31].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Identifying the protein region that mediates drug sensitivity established that TOP2 is the physical target of anticancer poisons and localized the drug-interaction surface.\",\n      \"evidence\": \"purification and in vitro cleavage assays of the yeast top2-5 mutant enzyme with amsacrine and etoposide\",\n      \"pmids\": [\"8395511\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve atomic structure of the drug-binding pocket\", \"Yeast enzyme, not human TOP2A\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Mapping Ser741 as a shared quinolone/etoposide contact showed how single residues control ternary cleavage-complex stability and drug reversibility.\",\n      \"evidence\": \"site-directed mutagenesis and in vitro cleavage assays with etoposide and quinolone CP-115,953\",\n      \"pmids\": [\"7657608\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of complex irreversibility at the structural level unresolved\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Cross-species complementation answered whether human TOP2A's specialized roles arise from catalysis itself, showing both human isoforms are catalytically interchangeable and isozyme specificity is extrinsic.\",\n      \"evidence\": \"complementation of yeast top2 mutants with human TOP2A and TOP2B constructs across multiple phenotypes\",\n      \"pmids\": [\"8804406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the extrinsic factors conferring isozyme specificity\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating Tdp1 acts on 5'-phosphotyrosyl Top2 adducts opened the question of how covalent TOP2-DNA lesions are repaired.\",\n      \"evidence\": \"yeast deletion analysis plus in vitro biochemistry on Top2 peptide-DNA substrates and drug-sensitivity epistasis\",\n      \"pmids\": [\"16751265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish requirement for prior proteolytic processing\", \"Relative contribution versus other phosphodiesterases unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Genomic mapping at replication forks defined where and when Top2 relieves torsional stress, establishing its coordinated action with Top1 to prevent fork-associated breakage.\",\n      \"evidence\": \"ChIP-on-chip of Top1/Top2 on replicating yeast chromosomes with top1/top2 epistasis and Rad53 checkpoint assays\",\n      \"pmids\": [\"17671091\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of sister chromatid junction accumulation not fully resolved\", \"Yeast system\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linking Top2 occupancy to transcription-associated fragile loci and identifying Bmi1/Ring1A as the E3 ligase for drug-poisoned TOP2A connected enzyme localization and degradation to genome stability and drug efficacy.\",\n      \"evidence\": \"ChIP-chip of Top2/Hmo1 with γH2A mapping; siRNA screen plus in vitro ubiquitination and small-molecule ligase inhibition\",\n      \"pmids\": [\"19737516\", \"19956605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ubiquitination directly precedes TDP2 action not yet tested\", \"Bmi1 substrate specificity for TOP2A versus other targets\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defining the HuR/miR-548c-3p competition at the 3'-UTR and the continuous mitotic requirement for Top2 established translational control of TOP2A abundance and its persistent role through telophase.\",\n      \"evidence\": \"RNA-IP, MS2-tagged RNA precipitation and reporter assays; yeast temperature-shift epistasis distinguishing Top2 from condensin\",\n      \"pmids\": [\"21768308\", \"21540296\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Medium confidence for the mitotic-timing finding\", \"How translational control couples to cell-cycle demand unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showing irreversible Top2cc must be proteolytically processed before TDP2 can act, plus a TDP2 crystal structure, defined the ordered processing of trapped complexes and TDP2 substrate accommodation.\",\n      \"evidence\": \"in vitro Top2cc processing with suicide substrates, TDP2 enzymatic assays, and a 1.6 Å crystal structure\",\n      \"pmids\": [\"24808172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the in vivo protease(s) not defined here\", \"Coupling to cellular degradation machinery unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identifying TOP2-BAF synergy, PTEN/OTUD3-mediated stabilization, Top2/Sgs1-Top3 redundancy, and TDP1 involvement broadened TOP2A function from torsional relief to catenation-based chromatin remodeling, checkpoint control, and break repair.\",\n      \"evidence\": \"genome-wide ATAC-seq with inhibitors; PTEN/TOP2A co-IP and decatenation checkpoint assays; yeast 2D-gel epistasis; TDP1 knockdown with cytogenetics\",\n      \"pmids\": [\"28250416\", \"26657567\", \"26630413\", \"26421495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PTEN/OTUD3 and TDP1 findings are Medium confidence and lack in vitro reconstitution\", \"Mechanism by which catenation state distinguishes heterochromatin from open chromatin unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrating replication-independent formation and condensin-biased anaphase removal of sister chromatid intertwines resolved how Top2 toggles between concatenation and decatenation during the cell cycle.\",\n      \"evidence\": \"yeast genetics, 2D gels for SCIs, and condensin/Top2 conditional mutants across cell-cycle stages\",\n      \"pmids\": [\"27716481\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular signal that biases Top2 toward decatenation not fully defined\", \"Yeast system\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Single-base cleavage mapping, G4-ligand and CX-5461 cytotoxicity dependence, and Rrp2-mediated protection against SUMO-dependent degradation linked TOP2A genomic targeting and stability to drug responses and concealed-break exposure.\",\n      \"evidence\": \"genome-wide cleavage sequencing in K562; unbiased genetic screens with G4 ligands; CRISPR/deletion screen with SUMO-binding biochemistry in fission yeast\",\n      \"pmids\": [\"28385713\", \"34180392\", \"28552615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of CTCF-proximal versus transcription-proximal cleavage partitioning unresolved\", \"Human ortholog of Rrp2 function not established here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showing TDP2 cannot act on intact Top2ccs and identifying a dominant-negative TOP2A/90 isoform clarified that processing precedes TDP2 and that splice isoforms drive chemoresistance.\",\n      \"evidence\": \"TARDIS assays with TDP2 knockdown; co-IP and bidirectional gain/loss of function for TOP2α/90\",\n      \"pmids\": [\"30011940\", \"29514855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TDP2-processing finding is Medium confidence\", \"Physiological abundance and regulation of TOP2α/90 isoform unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defining MDM4 binding and CTCF/transcription-proximal cleavage distribution connected TOP2A to p53 regulation and refined its genomic break landscape relative to chromatin features.\",\n      \"evidence\": \"co-IP with domain mapping and p53 activity assays; strand-specific nucleotide-resolution cleavage mapping in yeast and human\",\n      \"pmids\": [\"30672125\", \"31649282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MDM4 finding is Medium confidence with no in vitro reconstitution\", \"Functional consequence of SSB-prone sites in vivo not fully defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing the ZATT-TOP2A-PICH fork-reversal axis, ubiquitin-required adduct liberation, anthracycline poison-to-inhibitor switching, and TOP2α-dependent T cell apoptosis integrated TOP2A SUMOylation, ubiquitination, and drug pharmacology into genome-protective and pathological pathways.\",\n      \"evidence\": \"co-IP, PLA, fork-reversal EM and SUMOylation assays; TARDIS with ubiquitin-activating enzyme inhibitors; in vitro cleavage with isoform-specific iPSC-cardiomyocyte assays; enzymatic activity and apoptosis assays in primary T cells\",\n      \"pmids\": [\"33296677\", \"32587095\", \"31399497\", \"32193368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination and T-cell findings are Medium confidence\", \"How SUMOylation status routes between PICH recruitment and degradation unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying VCP/p97 as required for proteasomal degradation of trapped complexes placed an AAA-ATPase upstream of break exposure in adduct resolution.\",\n      \"evidence\": \"TARDIS assays with VCP/p97 inhibition, proteasome epistasis, and γH2AX readout\",\n      \"pmids\": [\"33941661\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without reconstitution\", \"How VCP/p97 recognizes TOP2-DNA adducts not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Establishing TOP2A requirements in trophoblast, decidualization, neurodevelopment, and its E2F1/EZH2/USP15 regulatory wiring extended TOP2A function to developmental programs and tissue-specific transcriptional control.\",\n      \"evidence\": \"shRNA knockdown with pathway profiling and embryo/mouse models; ChIP and reporter assays for E2F1; zebrafish/mouse behavior with H3K27me3 ChIP; co-IP with USP15-dependent stability assays\",\n      \"pmids\": [\"36585615\", \"36138481\", \"36417527\", \"36550695\", \"36065190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"All findings Medium confidence in single labs\", \"Whether developmental phenotypes reflect catalytic topoisomerase activity or non-catalytic roles is unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defining a cytokinetic abscission checkpoint driven by Top2cc degradation, RAD54L2-mediated SUMO-dependent resolution, and an EZH2/miR-139-5p axis connected TOP2A adduct handling to mitotic checkpoint signaling and tumor proliferation.\",\n      \"evidence\": \"live imaging and IF for the Rad17-MRN-ATM-Chk2-INCENP pathway; co-IP/ATPase and TDP2/ZATT epistasis for RAD54L2; ChIP, reporter, and senescence/xenograft assays for EZH2/miR-139-5p\",\n      \"pmids\": [\"37638884\", \"38055822\", \"38008711\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RAD54L2 and EZH2 findings are Medium confidence\", \"Relationship between TDP2-independent and TDP2-dependent resolution pathways in vivo not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying TOP2α-dependent rDNA-promoter damage from CX-5461 and a KDM5B/ZBTB16 axis controlling TOP2A in chemoresistance extended TOP2A targeting and regulation to nucleolar damage and epigenetic drug-resistance circuits.\",\n      \"evidence\": \"RADAR/Top2cc assays with genetic dependency in Eµ-Myc lymphoma; ChIP for H3K4me3 with USP7 ubiquitination and cisplatin sensitivity assays\",\n      \"pmids\": [\"39062087\", \"38287116\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Both findings Medium confidence in single labs\", \"Generality of rDNA-localized TOP2α damage across cell types unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved which proteases and how the choice between proteasomal versus SUMO-dependent (ZATT/RAD54L2) resolution routes is made for trapped TOP2A-DNA complexes, and what extrinsic factors confer TOP2A isozyme specificity in human cells.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo protease(s) for Top2cc processing unidentified\", \"Determinants routing SUMOylated TOP2A to PICH recruitment versus degradation undefined\", \"Extrinsic isozyme-specificity factors not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0, 6, 13, 19]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 6, 17]},\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [5, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3, 6, 25]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [13, 14, 15]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [30]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [9, 13, 25]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [0, 7, 35]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 2, 23, 26]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [5, 29]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [12, 20, 33]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PICH\", \"ZATT\", \"PTEN\", \"OTUD3\", \"MDM4\", \"GINS1\", \"RAD54L2\", \"TDP2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":9,"faith_pct":88.88888888888889}}