Affinage

EME2

Structure-specific endonuclease subunit EME2 · UniProt A4GXA9

Length
379 aa
Mass
41.2 kDa
Annotated
2026-06-09
19 papers in source corpus 10 papers cited in narrative 10 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 2012 Medium

    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

    PMID:22551069

    Open questions at the time
    • Does not distinguish EME1- versus EME2-containing complexes in FEN1 stimulation
    • Single-lab cellular co-localization without functional consequence defined
  2. 2013 High

    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

    PMID:24371268

    Open questions at the time
    • In vitro substrate preferences did not yet establish the in vivo substrate
    • Cell-cycle context of activity not addressed
  3. 2013 High

    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

    PMID:23982516

    Open questions at the time
    • Full-complex architecture not resolved here
    • No EME2-specific structural role distinguished
  4. 2014 High

    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

    PMID:24813886

    Open questions at the time
    • Mechanism enforcing temporal specificity not yet defined
    • Direct in vivo substrate at forks inferred from phenotype
  5. 2014 High

    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

    PMID:24692662

    Open questions at the time
    • Physiological relevance of intact HJ cleavage by the S-phase complex unclear
    • No structural basis for substrate selection yet
  6. 2016 High

    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

    PMID:26804904

    Open questions at the time
    • Precise fork structures cleaved in vivo not directly visualized
    • Does not address how MUS81-EME2 is activated upon Chk1 loss
  7. 2016 High

    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

    PMID:27997828

    Open questions at the time
    • Direct phosphorylation sites controlling EME2-containing complex assembly not mapped
    • How EME2 is selected over EME1/SLX4 during S phase not fully resolved
  8. 2018 Medium

    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

    PMID:29777036

    Open questions at the time
    • Single-lab study
    • Direct cleavage of Myc-induced stalled forks inferred from synthetic phenotype
  9. 2022 High

    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

    PMID:35290797

    Open questions at the time
    • Does not explain how identical enzymes are differentially recruited in cells
    • EME2-specific regulatory interactions not captured in the structure
  10. 2023 Medium

    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

    PMID:37352577

    Open questions at the time
    • Inhibitor is not selective between EME1 and EME2 complexes
    • Cellular specificity and off-target effects not fully characterized

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • EME2-specific recruitment/activation mechanism unknown
  • In vivo fork substrates not directly visualized
  • Structural basis for any EME2-specific regulation undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140097 catalytic activity, acting on DNA 3 GO:0098772 molecular function regulator activity 2 GO:0003677 DNA binding 1
Pathway
R-HSA-73894 DNA Repair 4 R-HSA-69306 DNA Replication 2
Partners
FEN1MUS81
Complex memberships
MUS81-EME2

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2013 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. Purified recombinant protein in vitro endonuclease assays comparing MUS81-EME1 and MUS81-EME2 on defined DNA substrates Nucleic acids research High 24371268
2014 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. siRNA knockdown of EME1 or EME2 in human cells with fork restart assays, DNA fiber analysis, and ALT telomere length assays Cell reports High 24813886
2014 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. In vitro endonuclease assays with recombinant MUS81-EME2 purified from E. coli on defined DNA substrates including intact HJs, nicked and gapped duplexes Nucleic acids research High 24692662
2013 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. 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 Nucleic acids research High 23982516
2012 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. Co-immunoprecipitation, Michaelis-Menten kinetic analysis of FEN1 stimulation by MUS81, truncation mapping, immunofluorescence co-localization in DNA-damaged human cells The FEBS journal Medium 22551069
2016 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. Genetic epistasis: siRNA/shRNA co-depletion of Chk1 with Mus81-Eme2 or Mre11, DNA fiber analysis, γH2AX detection, and ATM pathway inhibition Cell reports High 26804904
2016 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. Cell-cycle-stage-specific complex immunoprecipitation, WEE1 inhibitor treatment, CDK1/PLK1 manipulation, chromosome pulverization assays, epistasis with MUS81 depletion Developmental cell High 27997828
2018 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. siRNA co-depletion of MUS81-EME2 and Polη in Myc-overexpressing cells; γH2AX quantification, DNA fiber analysis, cell viability assays Journal of cell science Medium 29777036
2022 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. X-ray crystallography of MUS81-EME2 complex; structure-guided mutagenesis; in vitro endonuclease activity assays on multiple DNA substrates Structure (London, England : 1993) High 35290797
2023 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. FRET-based high-throughput endonuclease assay screening, surface plasmon resonance, EMSA, γH2AX immunofluorescence in HeLa cells Bioorganic & medicinal chemistry Medium 37352577

Source papers

Stage 0 corpus · 19 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2016 A Mechanism for Controlled Breakage of Under-replicated Chromosomes during Mitosis. Developmental cell 108 27997828
2014 MUS81-EME2 promotes replication fork restart. Cell reports 107 24813886
2016 Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage. Cell reports 65 26804904
2013 Substrate specificity of the MUS81-EME2 structure selective endonuclease. Nucleic acids research 49 24371268
2021 The plasma peptides of Alzheimer's disease. Clinical proteomics 33 34182925
2014 Human MUS81-EME2 can cleave a variety of DNA structures including intact Holliday junction and nicked duplex. Nucleic acids research 30 24692662
2018 Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress. Journal of cell science 25 29777036
2013 A winged helix domain in human MUS81 binds DNA and modulates the endonuclease activity of MUS81 complexes. Nucleic acids research 12 23982516
2012 Human MUS81 complexes stimulate flap endonuclease 1. The FEBS journal 11 22551069
2010 Gamma-radiation sensitivity and polymorphisms in RAD51L1 modulate glioma risk. Carcinogenesis 11 20610542
2022 Comprehensive Analysis of Cell Cycle-Related Genes in Patients With Prostate Cancer. Frontiers in oncology 9 35087757
2022 Crystal structure of the human MUS81-EME2 complex. Structure (London, England : 1993) 8 35290797
2020 Investigation of the immunogenicity of Zika glycan loop. Virology journal 8 32234060
2023 Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening. Bioorganic & medicinal chemistry 7 37352577
2023 A Novel Four Mitochondrial Respiration-Related Signature for Predicting Biochemical Recurrence of Prostate Cancer. Journal of clinical medicine 6 36675580
2020 Identification of a Splenic Marginal Zone Lymphoma Signature: Preliminary Findings With Diagnostic Potential. Frontiers in oncology 6 32457837
2022 A protocol to determine the activities of human MUS81-EME1&2 endonucleases. STAR protocols 3 35819885
2024 Novel molecular typing reveals the risk of recurrence in patients with early-stage papillary thyroid cancer. Thyroid research 1 38556856
2026 Whole exome sequencing reveals rare DNA repair gene variants in BRCA1/2-negative Arab early-onset breast cancer patients. Scientific reports 0 42098349

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