{"gene":"EME2","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2013,"finding":"MUS81-EME2 is a structure-selective endonuclease with broader substrate specificity than MUS81-EME1: it cleaves 3'-flaps, replication forks, nicked Holliday junctions, D-loop recombination intermediates (by cleaving the 3'-invading strand), and 5'-flap structures in reactions that MUS81-EME1 cannot promote. MUS81-EME2 is overall a more active endonuclease than MUS81-EME1.","method":"Purified recombinant protein in vitro endonuclease assays comparing MUS81-EME1 and MUS81-EME2 on defined DNA substrates","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins and multiple substrate assays, replicated in independent study (PMID:24692662)","pmids":["24371268"],"is_preprint":false},{"year":2014,"finding":"MUS81-EME2 is the S-phase-specific endonuclease responsible for replication fork cleavage and restart in human cells, and is also responsible for telomere maintenance in telomerase-negative ALT cells. In contrast, G2/M functions of MUS81 (cleavage of recombination intermediates, fragile site expression) are promoted by MUS81-EME1, defining temporally distinct roles for the two complexes.","method":"siRNA knockdown of EME1 or EME2 in human cells with fork restart assays, DNA fiber analysis, and ALT telomere length assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockdown with defined cellular phenotypes using multiple orthogonal readouts, replicated conceptually by other studies","pmids":["24813886"],"is_preprint":false},{"year":2014,"finding":"Human recombinant MUS81-EME2 cleaves intact Holliday junctions relatively efficiently compared to MUS81-EME1, and also catalyzes cleavage of nicked and gapped duplex DNAs generating double-strand breaks. The presence of a 5' phosphate at nicks renders DNA significantly less susceptible to cleavage by MUS81-EME2.","method":"In vitro endonuclease assays with recombinant MUS81-EME2 purified from E. coli on defined DNA substrates including intact HJs, nicked and gapped duplexes","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified protein, multiple substrate types tested, independent replication of broader substrate specificity finding","pmids":["24692662"],"is_preprint":false},{"year":2013,"finding":"MUS81 contains a winged helix (WH) domain that binds DNA and modulates endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes. WH domain mutations reduce DNA binding and incision activity, and deletion of the WH domain reduces endonuclease activity of MUS81-EME2; incisions by MUS81-EME2 are made closer to the junction on fork and nicked HJ substrates when the WH domain is deleted.","method":"Crystal structure of WH domain; WH domain mutagenesis combined with in vitro DNA binding and endonuclease activity assays with MUS81-EME1/EME2 complexes; S. pombe genetic complementation","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus in vitro functional assays, multiple orthogonal methods in single study","pmids":["23982516"],"is_preprint":false},{"year":2012,"finding":"Both MUS81-EME1 and MUS81-EME2 stimulate FEN1 (flap endonuclease 1) endonuclease activity, but FEN1 does not reciprocally stimulate MUS81-EME1 or MUS81-EME2. The MUS81 subunit alone (and its N-terminal half) is sufficient to bind FEN1 and stimulate its activity; MUS81 increases FEN1-substrate interaction, raising turnover. After DNA damage, FEN1 co-localizes with MUS81 in human cells.","method":"Co-immunoprecipitation, Michaelis-Menten kinetic analysis of FEN1 stimulation by MUS81, truncation mapping, immunofluorescence co-localization in DNA-damaged human cells","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical assays with kinetics and domain mapping plus cellular co-localization, single lab","pmids":["22551069"],"is_preprint":false},{"year":2016,"finding":"In Chk1-deficient cells, MUS81-EME2 (the S-phase nuclease complex) generates DNA damage that triggers ATM-dependent DDR signaling, which in turn causes replication fork slowing and increased origin firing. Genetic invalidation of Mus81-Eme2 suppresses both the DNA damage and the replication phenotypes of Chk1-deficient cells, placing MUS81-EME2 as the nuclease upstream of DDR-mediated replication dynamics modulation.","method":"Genetic epistasis: siRNA/shRNA co-depletion of Chk1 with Mus81-Eme2 or Mre11, DNA fiber analysis, γH2AX detection, and ATM pathway inhibition","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple knockdown combinations and multiple phenotypic readouts, mechanistic pathway placement","pmids":["26804904"],"is_preprint":false},{"year":2016,"finding":"MUS81-SLX4 assembly (activating MUS81 for mitotic cleavage of replication intermediates) is restrained during S phase by WEE1 kinase, which limits CDK1 and PLK1-mediated complex formation. MUS81-EME2 (S-phase form) and MUS81-SLX4 (M-phase form) thus constitute a double-negative feedback loop that renders replication and mitosis mutually exclusive. MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates to safeguard chromosome segregation.","method":"Cell-cycle-stage-specific complex immunoprecipitation, WEE1 inhibitor treatment, CDK1/PLK1 manipulation, chromosome pulverization assays, epistasis with MUS81 depletion","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP of complexes at defined cell cycle stages, kinase inhibition, genetic epistasis, imaging of chromosome phenotypes), mechanistic pathway placement","pmids":["27997828"],"is_preprint":false},{"year":2018,"finding":"MUS81-EME2 (the S-phase-specific endonuclease) generates DSBs at Myc-induced replication stress sites in the absence of Polη. Concomitant depletion of MUS81-EME2 and Polη synergistically enhances replication stress and cell death, indicating that MUS81-EME2 cleaves stalled forks during Myc-induced replication stress when TLS is compromised.","method":"siRNA co-depletion of MUS81-EME2 and Polη in Myc-overexpressing cells; γH2AX quantification, DNA fiber analysis, cell viability assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined phenotypic readouts, single lab, consistent with established S-phase role","pmids":["29777036"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of the human MUS81-EME2 complex reveals an overall protein fold similar to MUS81-EME1. Structure-guided mutagenesis shows MUS81-EME1 and MUS81-EME2 are identical in substrate recognition and endonuclease activities in vitro, implying their distinct cellular roles arise from temporal (cell-cycle) control rather than intrinsic biochemical differences.","method":"X-ray crystallography of MUS81-EME2 complex; structure-guided mutagenesis; in vitro endonuclease activity assays on multiple DNA substrates","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with extensive mutagenesis and biochemical validation, single rigorous study with multiple orthogonal methods","pmids":["35290797"],"is_preprint":false},{"year":2023,"finding":"Dyngo-4a is a small-molecule inhibitor of both MUS81-EME1 and MUS81-EME2 complexes (IC50 0.57 μM and 2.90 μM, respectively). It directly binds MUS81 complexes (KD ~0.61 μM by SPR) and prevents them from binding DNA substrates (shown by EMSA). In HeLa cells, dyngo-4a suppresses bleomycin-triggered γH2AX.","method":"FRET-based high-throughput endonuclease assay screening, surface plasmon resonance, EMSA, γH2AX immunofluorescence in HeLa cells","journal":"Bioorganic & medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro binding and activity assays with direct biochemical characterization plus cellular validation, single lab","pmids":["37352577"],"is_preprint":false}],"current_model":"EME2 is the regulatory subunit of the S-phase-specific human MUS81-EME2 structure-selective endonuclease complex, which cleaves stalled/collapsed replication forks (including D-loops, 5'-flaps, nicked HJs, and intact HJs) to promote fork restart and telomere maintenance in ALT cells; its activity during S phase is temporally separated from the MUS81-EME1/SLX4 complex (which acts in G2/M) through WEE1-CDK1-PLK1 control of complex assembly, and structurally the MUS81-EME2 crystal structure shows a fold similar to MUS81-EME1 with a key WH domain in MUS81 required for DNA binding and proper incision positioning."},"narrative":{"mechanistic_narrative":"EME2 is the regulatory subunit of the human MUS81-EME2 structure-selective endonuclease, an S-phase-specific complex that resolves stalled and collapsed replication intermediates to enable fork restart [PMID:24813886]. Reconstituted MUS81-EME2 cleaves a broad range of branched DNA substrates—3'-flaps, replication forks, nicked Holliday junctions, D-loop recombination intermediates (incising the 3'-invading strand), 5'-flaps, and intact Holliday junctions, as well as nicked and gapped duplexes to generate double-strand breaks—and is overall a more active nuclease than MUS81-EME1 [PMID:24371268, PMID:24692662]. A winged-helix domain in the MUS81 subunit binds DNA and positions the incision relative to the junction [PMID:23982516], and the MUS81-EME2 crystal structure shows a fold essentially identical to MUS81-EME1 with indistinguishable in vitro substrate recognition, establishing that the distinct cellular roles of the two complexes arise from temporal control rather than intrinsic biochemistry [PMID:35290797]. This temporal separation is enforced by WEE1, which restrains CDK1/PLK1-driven MUS81-SLX4 assembly during S phase, creating a double-negative feedback loop that confines MUS81-EME2 to replication and MUS81-SLX4/EME1 to mitosis [PMID:27997828]. Functionally, MUS81-EME2 maintains telomeres in telomerase-negative ALT cells [PMID:24813886] and acts as the nuclease that cleaves stalled forks under oncogenic and checkpoint stress: it generates the ATM-activating DNA damage that drives replication fork slowing and origin firing in Chk1-deficient cells [PMID:26804904], and produces double-strand breaks at Myc-induced replication stress sites when translesion synthesis by Polη is compromised [PMID:29777036].","teleology":[{"year":2012,"claim":"Established a physical and functional link between MUS81 complexes and another flap nuclease, defining cooperative DNA-processing rather than isolated activity.","evidence":"Co-IP, kinetic analysis, truncation mapping and damage-induced co-localization showing MUS81 binds and stimulates FEN1","pmids":["22551069"],"confidence":"Medium","gaps":["Does not distinguish EME1- versus EME2-containing complexes in FEN1 stimulation","Single-lab cellular co-localization without functional consequence defined"]},{"year":2013,"claim":"Defined the substrate repertoire of MUS81-EME2, showing it is biochemically broader and more active than MUS81-EME1.","evidence":"In vitro endonuclease assays with purified recombinant complexes on defined branched DNA substrates","pmids":["24371268"],"confidence":"High","gaps":["In vitro substrate preferences did not yet establish the in vivo substrate","Cell-cycle context of activity not addressed"]},{"year":2013,"claim":"Identified the structural element in MUS81 (the winged-helix domain) that controls DNA binding and incision positioning for both complexes.","evidence":"WH-domain crystal structure with mutagenesis, in vitro binding/cleavage assays, and S. pombe complementation","pmids":["23982516"],"confidence":"High","gaps":["Full-complex architecture not resolved here","No EME2-specific structural role distinguished"]},{"year":2014,"claim":"Resolved the cellular division of labor, assigning MUS81-EME2 the S-phase fork-cleavage/restart and ALT telomere maintenance roles distinct from the G2/M roles of MUS81-EME1.","evidence":"siRNA knockdown of EME1 vs EME2 with fork-restart assays, DNA fiber analysis and ALT telomere measurements","pmids":["24813886"],"confidence":"High","gaps":["Mechanism enforcing temporal specificity not yet defined","Direct in vivo substrate at forks inferred from phenotype"]},{"year":2014,"claim":"Confirmed and extended the broad substrate specificity, including efficient intact Holliday junction cleavage and double-strand break generation from nicked/gapped duplexes.","evidence":"In vitro endonuclease assays with E. coli-purified recombinant MUS81-EME2 on multiple substrate types","pmids":["24692662"],"confidence":"High","gaps":["Physiological relevance of intact HJ cleavage by the S-phase complex unclear","No structural basis for substrate selection yet"]},{"year":2016,"claim":"Placed MUS81-EME2 upstream of the DNA damage response in checkpoint-deficient cells, showing its cleavage drives replication dynamics rather than being a passive consequence of damage.","evidence":"Genetic epistasis co-depleting Chk1 with Mus81-Eme2 or Mre11, DNA fiber analysis, γH2AX, and ATM inhibition","pmids":["26804904"],"confidence":"High","gaps":["Precise fork structures cleaved in vivo not directly visualized","Does not address how MUS81-EME2 is activated upon Chk1 loss"]},{"year":2016,"claim":"Identified the kinase circuit (WEE1-CDK1-PLK1) that temporally segregates the S-phase MUS81-EME2 form from the mitotic MUS81-SLX4 form via a double-negative feedback loop.","evidence":"Cell-cycle-stage complex Co-IP, WEE1 inhibition, CDK1/PLK1 manipulation, chromosome pulverization assays and MUS81-depletion epistasis","pmids":["27997828"],"confidence":"High","gaps":["Direct phosphorylation sites controlling EME2-containing complex assembly not mapped","How EME2 is selected over EME1/SLX4 during S phase not fully resolved"]},{"year":2018,"claim":"Demonstrated that MUS81-EME2 cleaves stalled forks under oncogene-induced replication stress when translesion synthesis is unavailable, defining a backup fork-processing role.","evidence":"siRNA co-depletion of MUS81-EME2 and Polη in Myc-overexpressing cells with γH2AX, DNA fiber, and viability assays","pmids":["29777036"],"confidence":"Medium","gaps":["Single-lab study","Direct cleavage of Myc-induced stalled forks inferred from synthetic phenotype"]},{"year":2022,"claim":"Solved the MUS81-EME2 structure and showed it is biochemically indistinguishable from MUS81-EME1, establishing that functional divergence is temporal, not intrinsic.","evidence":"X-ray crystallography of the human MUS81-EME2 complex with structure-guided mutagenesis and in vitro endonuclease assays","pmids":["35290797"],"confidence":"High","gaps":["Does not explain how identical enzymes are differentially recruited in cells","EME2-specific regulatory interactions not captured in the structure"]},{"year":2023,"claim":"Provided a chemical tool (dyngo-4a) that directly binds MUS81 complexes and blocks DNA engagement, enabling pharmacological probing of MUS81-EME2 activity.","evidence":"FRET endonuclease screening, SPR binding, EMSA, and γH2AX suppression in HeLa cells","pmids":["37352577"],"confidence":"Medium","gaps":["Inhibitor is not selective between EME1 and EME2 complexes","Cellular specificity and off-target effects not fully characterized"]},{"year":null,"claim":"How a biochemically identical enzyme is selectively recruited to and activated at specific replication structures during S phase, and the molecular determinants distinguishing EME2 from EME1 in vivo, remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["EME2-specific recruitment/activation mechanism unknown","In vivo fork substrates not directly visualized","Structural basis for any EME2-specific regulation undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0,2,8]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,6]}],"localization":[],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,1,5,7]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[1,5]}],"complexes":["MUS81-EME2"],"partners":["MUS81","FEN1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"A4GXA9","full_name":"Structure-specific endonuclease subunit EME2","aliases":["Essential meiotic structure-specific endonuclease subunit 2"],"length_aa":379,"mass_kda":41.2,"function":"Non-catalytic subunit of the structure-specific, heterodimeric DNA endonuclease MUS81-EME2 which is involved in the maintenance of genome stability. In the complex, EME2 is required for DNA cleavage, participating in DNA recognition and bending (PubMed:17289582, PubMed:24371268, PubMed:24813886, PubMed:35290797). MUS81-EME2 cleaves 3'-flaps and nicked Holliday junctions, and exhibit limited endonuclease activity with 5' flaps and nicked double-stranded DNAs (PubMed:24371268). MUS81-EME2 which is active during the replication of DNA is more specifically involved in replication fork processing (PubMed:17289582, PubMed:24813886). Replication forks frequently encounter obstacles to their passage, including DNA base lesions, DNA interstrand cross-links, difficult-to-replicate sequences, transcription bubbles, or tightly bound proteins. One mechanism for the restart of a stalled replication fork involves nucleolytic cleavage mediated by the MUS81-EME2 endonuclease. By acting upon the stalled fork, MUS81-EME2 generates a DNA double-strand break (DSB) that can be repaired by homologous recombination, leading to the restoration of an active fork (PubMed:24813886). MUS81-EME2 could also function in telomere maintenance (PubMed:24813886)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/A4GXA9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EME2","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EME2","total_profiled":1310},"omim":[{"mim_id":"610886","title":"ESSENTIAL MEIOTIC STRUCTURE-SPECIFIC ENDONUCLEASE 2; EME2","url":"https://www.omim.org/entry/610886"},{"mim_id":"610885","title":"ESSENTIAL MEIOTIC STRUCTURE-SPECIFIC ENDONUCLEASE 1; EME1","url":"https://www.omim.org/entry/610885"},{"mim_id":"606591","title":"MUS81 STRUCTURE-SPECIFIC ENDONUCLEASE SUBUNIT; MUS81","url":"https://www.omim.org/entry/606591"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EME2"},"hgnc":{"alias_symbol":["FLJ00151","SLX2B"],"prev_symbol":[]},"alphafold":{"accession":"A4GXA9","domains":[{"cath_id":"3.40.50.10130","chopping":"59-196_214-266","consensus_level":"high","plddt":87.848,"start":59,"end":266},{"cath_id":"1.10.150.670","chopping":"289-368","consensus_level":"high","plddt":93.0359,"start":289,"end":368}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/A4GXA9","model_url":"https://alphafold.ebi.ac.uk/files/AF-A4GXA9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-A4GXA9-F1-predicted_aligned_error_v6.png","plddt_mean":79.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EME2","jax_strain_url":"https://www.jax.org/strain/search?query=EME2"},"sequence":{"accession":"A4GXA9","fasta_url":"https://rest.uniprot.org/uniprotkb/A4GXA9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/A4GXA9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/A4GXA9"}},"corpus_meta":[{"pmid":"27997828","id":"PMC_27997828","title":"A Mechanism for Controlled Breakage of Under-replicated Chromosomes during Mitosis.","date":"2016","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/27997828","citation_count":108,"is_preprint":false},{"pmid":"24813886","id":"PMC_24813886","title":"MUS81-EME2 promotes replication fork restart.","date":"2014","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/24813886","citation_count":107,"is_preprint":false},{"pmid":"26804904","id":"PMC_26804904","title":"Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage.","date":"2016","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/26804904","citation_count":65,"is_preprint":false},{"pmid":"24371268","id":"PMC_24371268","title":"Substrate specificity of the MUS81-EME2 structure selective endonuclease.","date":"2013","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/24371268","citation_count":49,"is_preprint":false},{"pmid":"34182925","id":"PMC_34182925","title":"The plasma peptides of Alzheimer's disease.","date":"2021","source":"Clinical proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/34182925","citation_count":33,"is_preprint":false},{"pmid":"24692662","id":"PMC_24692662","title":"Human MUS81-EME2 can cleave a variety of DNA structures including intact Holliday junction and nicked duplex.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/24692662","citation_count":30,"is_preprint":false},{"pmid":"29777036","id":"PMC_29777036","title":"Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress.","date":"2018","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/29777036","citation_count":25,"is_preprint":false},{"pmid":"23982516","id":"PMC_23982516","title":"A winged helix domain in human MUS81 binds DNA and modulates the endonuclease activity of MUS81 complexes.","date":"2013","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/23982516","citation_count":12,"is_preprint":false},{"pmid":"22551069","id":"PMC_22551069","title":"Human MUS81 complexes stimulate flap endonuclease 1.","date":"2012","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/22551069","citation_count":11,"is_preprint":false},{"pmid":"20610542","id":"PMC_20610542","title":"Gamma-radiation sensitivity and polymorphisms in RAD51L1 modulate glioma risk.","date":"2010","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/20610542","citation_count":11,"is_preprint":false},{"pmid":"35087757","id":"PMC_35087757","title":"Comprehensive Analysis of Cell Cycle-Related Genes in Patients With Prostate Cancer.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35087757","citation_count":9,"is_preprint":false},{"pmid":"35290797","id":"PMC_35290797","title":"Crystal structure of the human MUS81-EME2 complex.","date":"2022","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/35290797","citation_count":8,"is_preprint":false},{"pmid":"32234060","id":"PMC_32234060","title":"Investigation of the immunogenicity of Zika glycan loop.","date":"2020","source":"Virology journal","url":"https://pubmed.ncbi.nlm.nih.gov/32234060","citation_count":8,"is_preprint":false},{"pmid":"37352577","id":"PMC_37352577","title":"Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening.","date":"2023","source":"Bioorganic & medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37352577","citation_count":7,"is_preprint":false},{"pmid":"36675580","id":"PMC_36675580","title":"A Novel Four Mitochondrial Respiration-Related Signature for Predicting Biochemical Recurrence of Prostate Cancer.","date":"2023","source":"Journal of clinical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36675580","citation_count":6,"is_preprint":false},{"pmid":"32457837","id":"PMC_32457837","title":"Identification of a Splenic Marginal Zone Lymphoma Signature: Preliminary Findings With Diagnostic Potential.","date":"2020","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32457837","citation_count":6,"is_preprint":false},{"pmid":"35819885","id":"PMC_35819885","title":"A protocol to determine the activities of human MUS81-EME1&2 endonucleases.","date":"2022","source":"STAR protocols","url":"https://pubmed.ncbi.nlm.nih.gov/35819885","citation_count":3,"is_preprint":false},{"pmid":"38556856","id":"PMC_38556856","title":"Novel molecular typing reveals the risk of recurrence in patients with early-stage papillary thyroid cancer.","date":"2024","source":"Thyroid research","url":"https://pubmed.ncbi.nlm.nih.gov/38556856","citation_count":1,"is_preprint":false},{"pmid":"42098349","id":"PMC_42098349","title":"Whole exome sequencing reveals rare DNA repair gene variants in BRCA1/2-negative Arab early-onset breast cancer patients.","date":"2026","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/42098349","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.09.19.613851","title":"Potential Cancer Biomarkers: Mitotic Intra-S DNA Damage Checkpoint Genes","date":"2024-09-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.19.613851","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12199,"output_tokens":2931,"usd":0.040281,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10222,"output_tokens":3342,"usd":0.06733,"stage2_stop_reason":"end_turn"},"total_usd":0.107611,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"MUS81-EME2 is a structure-selective endonuclease with broader substrate specificity than MUS81-EME1: it cleaves 3'-flaps, replication forks, nicked Holliday junctions, D-loop recombination intermediates (by cleaving the 3'-invading strand), and 5'-flap structures in reactions that MUS81-EME1 cannot promote. MUS81-EME2 is overall a more active endonuclease than MUS81-EME1.\",\n      \"method\": \"Purified recombinant protein in vitro endonuclease assays comparing MUS81-EME1 and MUS81-EME2 on defined DNA substrates\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins and multiple substrate assays, replicated in independent study (PMID:24692662)\",\n      \"pmids\": [\"24371268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MUS81-EME2 is the S-phase-specific endonuclease responsible for replication fork cleavage and restart in human cells, and is also responsible for telomere maintenance in telomerase-negative ALT cells. In contrast, G2/M functions of MUS81 (cleavage of recombination intermediates, fragile site expression) are promoted by MUS81-EME1, defining temporally distinct roles for the two complexes.\",\n      \"method\": \"siRNA knockdown of EME1 or EME2 in human cells with fork restart assays, DNA fiber analysis, and ALT telomere length assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockdown with defined cellular phenotypes using multiple orthogonal readouts, replicated conceptually by other studies\",\n      \"pmids\": [\"24813886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Human recombinant MUS81-EME2 cleaves intact Holliday junctions relatively efficiently compared to MUS81-EME1, and also catalyzes cleavage of nicked and gapped duplex DNAs generating double-strand breaks. The presence of a 5' phosphate at nicks renders DNA significantly less susceptible to cleavage by MUS81-EME2.\",\n      \"method\": \"In vitro endonuclease assays with recombinant MUS81-EME2 purified from E. coli on defined DNA substrates including intact HJs, nicked and gapped duplexes\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified protein, multiple substrate types tested, independent replication of broader substrate specificity finding\",\n      \"pmids\": [\"24692662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MUS81 contains a winged helix (WH) domain that binds DNA and modulates endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes. WH domain mutations reduce DNA binding and incision activity, and deletion of the WH domain reduces endonuclease activity of MUS81-EME2; incisions by MUS81-EME2 are made closer to the junction on fork and nicked HJ substrates when the WH domain is deleted.\",\n      \"method\": \"Crystal structure of WH domain; WH domain mutagenesis combined with in vitro DNA binding and endonuclease activity assays with MUS81-EME1/EME2 complexes; S. pombe genetic complementation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus in vitro functional assays, multiple orthogonal methods in single study\",\n      \"pmids\": [\"23982516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Both MUS81-EME1 and MUS81-EME2 stimulate FEN1 (flap endonuclease 1) endonuclease activity, but FEN1 does not reciprocally stimulate MUS81-EME1 or MUS81-EME2. The MUS81 subunit alone (and its N-terminal half) is sufficient to bind FEN1 and stimulate its activity; MUS81 increases FEN1-substrate interaction, raising turnover. After DNA damage, FEN1 co-localizes with MUS81 in human cells.\",\n      \"method\": \"Co-immunoprecipitation, Michaelis-Menten kinetic analysis of FEN1 stimulation by MUS81, truncation mapping, immunofluorescence co-localization in DNA-damaged human cells\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical assays with kinetics and domain mapping plus cellular co-localization, single lab\",\n      \"pmids\": [\"22551069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In Chk1-deficient cells, MUS81-EME2 (the S-phase nuclease complex) generates DNA damage that triggers ATM-dependent DDR signaling, which in turn causes replication fork slowing and increased origin firing. Genetic invalidation of Mus81-Eme2 suppresses both the DNA damage and the replication phenotypes of Chk1-deficient cells, placing MUS81-EME2 as the nuclease upstream of DDR-mediated replication dynamics modulation.\",\n      \"method\": \"Genetic epistasis: siRNA/shRNA co-depletion of Chk1 with Mus81-Eme2 or Mre11, DNA fiber analysis, γH2AX detection, and ATM pathway inhibition\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple knockdown combinations and multiple phenotypic readouts, mechanistic pathway placement\",\n      \"pmids\": [\"26804904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MUS81-SLX4 assembly (activating MUS81 for mitotic cleavage of replication intermediates) is restrained during S phase by WEE1 kinase, which limits CDK1 and PLK1-mediated complex formation. MUS81-EME2 (S-phase form) and MUS81-SLX4 (M-phase form) thus constitute a double-negative feedback loop that renders replication and mitosis mutually exclusive. MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates to safeguard chromosome segregation.\",\n      \"method\": \"Cell-cycle-stage-specific complex immunoprecipitation, WEE1 inhibitor treatment, CDK1/PLK1 manipulation, chromosome pulverization assays, epistasis with MUS81 depletion\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP of complexes at defined cell cycle stages, kinase inhibition, genetic epistasis, imaging of chromosome phenotypes), mechanistic pathway placement\",\n      \"pmids\": [\"27997828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MUS81-EME2 (the S-phase-specific endonuclease) generates DSBs at Myc-induced replication stress sites in the absence of Polη. Concomitant depletion of MUS81-EME2 and Polη synergistically enhances replication stress and cell death, indicating that MUS81-EME2 cleaves stalled forks during Myc-induced replication stress when TLS is compromised.\",\n      \"method\": \"siRNA co-depletion of MUS81-EME2 and Polη in Myc-overexpressing cells; γH2AX quantification, DNA fiber analysis, cell viability assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined phenotypic readouts, single lab, consistent with established S-phase role\",\n      \"pmids\": [\"29777036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of the human MUS81-EME2 complex reveals an overall protein fold similar to MUS81-EME1. Structure-guided mutagenesis shows MUS81-EME1 and MUS81-EME2 are identical in substrate recognition and endonuclease activities in vitro, implying their distinct cellular roles arise from temporal (cell-cycle) control rather than intrinsic biochemical differences.\",\n      \"method\": \"X-ray crystallography of MUS81-EME2 complex; structure-guided mutagenesis; in vitro endonuclease activity assays on multiple DNA substrates\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with extensive mutagenesis and biochemical validation, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"35290797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Dyngo-4a is a small-molecule inhibitor of both MUS81-EME1 and MUS81-EME2 complexes (IC50 0.57 μM and 2.90 μM, respectively). It directly binds MUS81 complexes (KD ~0.61 μM by SPR) and prevents them from binding DNA substrates (shown by EMSA). In HeLa cells, dyngo-4a suppresses bleomycin-triggered γH2AX.\",\n      \"method\": \"FRET-based high-throughput endonuclease assay screening, surface plasmon resonance, EMSA, γH2AX immunofluorescence in HeLa cells\",\n      \"journal\": \"Bioorganic & medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro binding and activity assays with direct biochemical characterization plus cellular validation, single lab\",\n      \"pmids\": [\"37352577\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EME2 is the regulatory subunit of the S-phase-specific human MUS81-EME2 structure-selective endonuclease complex, which cleaves stalled/collapsed replication forks (including D-loops, 5'-flaps, nicked HJs, and intact HJs) to promote fork restart and telomere maintenance in ALT cells; its activity during S phase is temporally separated from the MUS81-EME1/SLX4 complex (which acts in G2/M) through WEE1-CDK1-PLK1 control of complex assembly, and structurally the MUS81-EME2 crystal structure shows a fold similar to MUS81-EME1 with a key WH domain in MUS81 required for DNA binding and proper incision positioning.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EME2 is the regulatory subunit of the human MUS81-EME2 structure-selective endonuclease, an S-phase-specific complex that resolves stalled and collapsed replication intermediates to enable fork restart [#1]. Reconstituted MUS81-EME2 cleaves a broad range of branched DNA substrates—3'-flaps, replication forks, nicked Holliday junctions, D-loop recombination intermediates (incising the 3'-invading strand), 5'-flaps, and intact Holliday junctions, as well as nicked and gapped duplexes to generate double-strand breaks—and is overall a more active nuclease than MUS81-EME1 [#0, #2]. A winged-helix domain in the MUS81 subunit binds DNA and positions the incision relative to the junction [#3], and the MUS81-EME2 crystal structure shows a fold essentially identical to MUS81-EME1 with indistinguishable in vitro substrate recognition, establishing that the distinct cellular roles of the two complexes arise from temporal control rather than intrinsic biochemistry [#8]. This temporal separation is enforced by WEE1, which restrains CDK1/PLK1-driven MUS81-SLX4 assembly during S phase, creating a double-negative feedback loop that confines MUS81-EME2 to replication and MUS81-SLX4/EME1 to mitosis [#6]. Functionally, MUS81-EME2 maintains telomeres in telomerase-negative ALT cells [#1] and acts as the nuclease that cleaves stalled forks under oncogenic and checkpoint stress: it generates the ATM-activating DNA damage that drives replication fork slowing and origin firing in Chk1-deficient cells [#5], and produces double-strand breaks at Myc-induced replication stress sites when translesion synthesis by Pol\\u03b7 is compromised [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established a physical and functional link between MUS81 complexes and another flap nuclease, defining cooperative DNA-processing rather than isolated activity.\",\n      \"evidence\": \"Co-IP, kinetic analysis, truncation mapping and damage-induced co-localization showing MUS81 binds and stimulates FEN1\",\n      \"pmids\": [\"22551069\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not distinguish EME1- versus EME2-containing complexes in FEN1 stimulation\", \"Single-lab cellular co-localization without functional consequence defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the substrate repertoire of MUS81-EME2, showing it is biochemically broader and more active than MUS81-EME1.\",\n      \"evidence\": \"In vitro endonuclease assays with purified recombinant complexes on defined branched DNA substrates\",\n      \"pmids\": [\"24371268\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro substrate preferences did not yet establish the in vivo substrate\", \"Cell-cycle context of activity not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified the structural element in MUS81 (the winged-helix domain) that controls DNA binding and incision positioning for both complexes.\",\n      \"evidence\": \"WH-domain crystal structure with mutagenesis, in vitro binding/cleavage assays, and S. pombe complementation\",\n      \"pmids\": [\"23982516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-complex architecture not resolved here\", \"No EME2-specific structural role distinguished\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the cellular division of labor, assigning MUS81-EME2 the S-phase fork-cleavage/restart and ALT telomere maintenance roles distinct from the G2/M roles of MUS81-EME1.\",\n      \"evidence\": \"siRNA knockdown of EME1 vs EME2 with fork-restart assays, DNA fiber analysis and ALT telomere measurements\",\n      \"pmids\": [\"24813886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism enforcing temporal specificity not yet defined\", \"Direct in vivo substrate at forks inferred from phenotype\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Confirmed and extended the broad substrate specificity, including efficient intact Holliday junction cleavage and double-strand break generation from nicked/gapped duplexes.\",\n      \"evidence\": \"In vitro endonuclease assays with E. coli-purified recombinant MUS81-EME2 on multiple substrate types\",\n      \"pmids\": [\"24692662\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of intact HJ cleavage by the S-phase complex unclear\", \"No structural basis for substrate selection yet\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed MUS81-EME2 upstream of the DNA damage response in checkpoint-deficient cells, showing its cleavage drives replication dynamics rather than being a passive consequence of damage.\",\n      \"evidence\": \"Genetic epistasis co-depleting Chk1 with Mus81-Eme2 or Mre11, DNA fiber analysis, \\u03b3H2AX, and ATM inhibition\",\n      \"pmids\": [\"26804904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise fork structures cleaved in vivo not directly visualized\", \"Does not address how MUS81-EME2 is activated upon Chk1 loss\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified the kinase circuit (WEE1-CDK1-PLK1) that temporally segregates the S-phase MUS81-EME2 form from the mitotic MUS81-SLX4 form via a double-negative feedback loop.\",\n      \"evidence\": \"Cell-cycle-stage complex Co-IP, WEE1 inhibition, CDK1/PLK1 manipulation, chromosome pulverization assays and MUS81-depletion epistasis\",\n      \"pmids\": [\"27997828\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphorylation sites controlling EME2-containing complex assembly not mapped\", \"How EME2 is selected over EME1/SLX4 during S phase not fully resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated that MUS81-EME2 cleaves stalled forks under oncogene-induced replication stress when translesion synthesis is unavailable, defining a backup fork-processing role.\",\n      \"evidence\": \"siRNA co-depletion of MUS81-EME2 and Pol\\u03b7 in Myc-overexpressing cells with \\u03b3H2AX, DNA fiber, and viability assays\",\n      \"pmids\": [\"29777036\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Direct cleavage of Myc-induced stalled forks inferred from synthetic phenotype\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Solved the MUS81-EME2 structure and showed it is biochemically indistinguishable from MUS81-EME1, establishing that functional divergence is temporal, not intrinsic.\",\n      \"evidence\": \"X-ray crystallography of the human MUS81-EME2 complex with structure-guided mutagenesis and in vitro endonuclease assays\",\n      \"pmids\": [\"35290797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not explain how identical enzymes are differentially recruited in cells\", \"EME2-specific regulatory interactions not captured in the structure\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided a chemical tool (dyngo-4a) that directly binds MUS81 complexes and blocks DNA engagement, enabling pharmacological probing of MUS81-EME2 activity.\",\n      \"evidence\": \"FRET endonuclease screening, SPR binding, EMSA, and \\u03b3H2AX suppression in HeLa cells\",\n      \"pmids\": [\"37352577\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Inhibitor is not selective between EME1 and EME2 complexes\", \"Cellular specificity and off-target effects not fully characterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a biochemically identical enzyme is selectively recruited to and activated at specific replication structures during S phase, and the molecular determinants distinguishing EME2 from EME1 in vivo, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"EME2-specific recruitment/activation mechanism unknown\", \"In vivo fork substrates not directly visualized\", \"Structural basis for any EME2-specific regulation undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0, 2, 8]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 1, 5, 7]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"complexes\": [\"MUS81-EME2\"],\n    \"partners\": [\"MUS81\", \"FEN1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}