{"gene":"CENPX","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2009,"finding":"CENP-X was identified as a new constitutive kinetochore protein forming a subcomplex with CENP-S. CENP-X-deficient chicken DT40 and human HeLa cells show abnormal mitotic behavior, and kinetochore localization of CENP-X requires CENP-T or CENP-K. Loss of CENP-X results in a significant reduction in outer kinetochore plate size and increased intrakinetochore distance, demonstrating its essential role in stable outer kinetochore assembly.","method":"Genetic knockouts in chicken DT40 cells, siRNA knockdown in HeLa cells, live cell imaging, electron microscopy of kinetochore outer plate","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal localization experiments, KO/KD in two cell systems with defined structural phenotype, live imaging","pmids":["19620631"],"is_preprint":false},{"year":2010,"finding":"MHF2 (CENP-X) and MHF1 (CENP-S) form a histone-fold-containing heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. Suppression of MHF1 destabilizes FANCM and MHF2, impairs FANCD2 monoubiquitination and foci formation, causes defective chromatin localization of FA core complex proteins, and increases MMC-induced chromosome aberrations.","method":"Co-immunoprecipitation, protein stability assays, DNA-binding biochemical assays, branch migration activity assay, siRNA knockdown with cellular phenotyping (immunofluorescence, MMC/CPT sensitivity)","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical reconstitution of DNA-binding and branch migration activity, combined with cellular loss-of-function phenotypes, replicated across multiple assays","pmids":["20347429"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of the MHF1-MHF2 (CENP-S/CENP-X) complex reveals that they form a compact tetramer. FANCM binds this tetramer through a 'dual-V' shaped structure, and FANCM-F together with (MHF1-MHF2)2 constitutes a new DNA-binding site coupled to the canonical L1L2 region. Perturbation of MHF-FANCM structural plasticity alters FANCM localization in vivo.","method":"X-ray crystallography of MHF1-MHF2 alone and in complex with FANCM fragment, in vivo localization assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure at atomic resolution with functional validation of FANCM localization in vivo","pmids":["22510687"],"is_preprint":false},{"year":2012,"finding":"The yeast MHF complex (Mhf1-Mhf2) adopts a histone-fold architecture structurally similar to the (H3-H4)2 heterotetramer. The heterotetrameric assembly is essential for the function of the complex in DNA repair, as shown by genetic data with interface mutants.","method":"X-ray crystallography, yeast genetics (mutant analysis of heterotetramer interfaces)","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with functional genetic validation in yeast","pmids":["22325783"],"is_preprint":false},{"year":2013,"finding":"The CENP-T-W-S-X complex binds preferentially to ~100 bp of linker DNA (not nucleosome-bound DNA), forms a (CENP-T-W-S-X)2 structure, and induces positive DNA supercoils in contrast to canonical nucleosomes. DNA-binding regions in CENP-T or CENP-W (but not CENP-S or CENP-X) are required for positive supercoiling activity and kinetochore targeting of the complex.","method":"DNA-binding assays, DNA supercoiling assay, mutagenesis of DNA-binding regions, kinetochore targeting assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and functional kinetochore targeting assays in a single rigorous study","pmids":["24234442"],"is_preprint":false},{"year":2013,"finding":"RSF1 facilitates the assembly of CENP-S and CENP-X at sites of DNA damage. Upon incorporation by RSF1, CENP-S and CENP-X promote assembly of the NHEJ factor XRCC4 at damaged chromatin, thereby promoting non-homologous end-joining. CENP-S and CENP-X are dispensable for homologous recombination.","method":"siRNA knockdown, laser microirradiation, immunofluorescence at DSB sites, NHEJ and HR repair assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi loss-of-function with defined DSB repair phenotypes and pathway placement, single lab","pmids":["23974106"],"is_preprint":false},{"year":2014,"finding":"RSF1, in an ATM-dependent manner, recruits CENP-X (MHF2) and CENP-S (MHF1) to DSB sites. CENP-X/MHF2 and CENP-S/MHF1 in turn regulate mono-ubiquitination of FANCD2 and FANCI at DSBs. The ATM-RSF1 interaction is dependent on DSBs and ATM kinase activity.","method":"Co-immunoprecipitation, laser microirradiation/immunofluorescence, siRNA knockdown, FANCD2/FANCI ubiquitination assays","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, RNAi with pathway-level phenotype, single lab","pmids":["24800743"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of human MHF1-MHF2 (CENP-S/CENP-X) bound to DNA reveals that MHF senses branched DNA by engaging two duplex arms simultaneously. Biochemical analyses confirm MHF preferentially binds DNA forks and four-way junctions over dsDNA. Mutations at the observed DNA-binding interface reduce cellular resistance to DNA damage.","method":"X-ray crystallography of MHF-DNA complex, DNA-binding biochemical assays, yeast genetic experiments with DNA-binding interface mutants","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with orthogonal biochemical validation and functional genetic confirmation","pmids":["24390579"],"is_preprint":false},{"year":2014,"finding":"CENP-S and CENP-X assemble de novo at centromeres during S phase and G2, increasing approximately 3-4 fold in abundance. FRET and fluorescence cross-correlation spectroscopy show that CENP-S and CENP-X exist in complex in soluble form and at centromeres. CENPX exchanges ~10 times faster than CENP-S at centromeres (t1/2 ~10 min vs ~120 min). CENP-T was identified as a strong CENP-S binding partner at centromeres by fluorescent two-hybrid and FRET, forming a CENP-S/X/T-containing complex near histone H3 but not CENP-A.","method":"Fluorescence cross-correlation spectroscopy, FRET, conditional labeling (EdU pulse-chase), FRAP, fluorescent two-hybrid assay","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal live-cell imaging methods (FRAP, FRET, FCCS) with quantitative functional readouts","pmids":["24522885"],"is_preprint":false},{"year":2013,"finding":"In fission yeast, Mhf1 and Mhf2 (CENP-S/CENP-X orthologs) perform two distinct functions: DNA repair/recombination (dependent on interaction with the FANCM ortholog Fml1) and centromere localization for chromosome segregation (independent of Fml1). Together with Fml1, they process sister chromatid junctions to aid chromosome segregation; the Mus81-Eme1 endonuclease acts as a failsafe for unresolved junctions.","method":"Fission yeast genetics (deletion mutants, epistasis), chromosome segregation assays, centromere localization experiments","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in model organism demonstrating two distinct functional pathways with defined molecular dependencies","pmids":["24026537"],"is_preprint":false},{"year":2013,"finding":"A SAXS study revealed that the (MHF1-MHF2)4 octamer presents a long, positively charged patch on its surface that plays a critical role in double-stranded DNA binding, providing the structural basis for anchoring the MHF-FANCM complex to chromatin.","method":"Small angle X-ray scattering (SAXS) in combination with crystallographic data","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural method (SAXS) in solution but single lab without mutagenesis validation of the identified patch","pmids":["23886707"],"is_preprint":false},{"year":2017,"finding":"In fission yeast, Mhf1/CENP-S and Mhf2/CENP-X histone-fold proteins together with Fml1/FANCM helicase suppress crossovers between centromere DNA repeats and prevent gross chromosomal rearrangements mediated by centromere repeats during mitosis.","method":"Fission yeast genetics (deletion mutants), recombination assays at centromere and non-centromere loci, chromosome rearrangement assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined recombination and chromosomal instability phenotypes at centromeres","pmids":["28977643"],"is_preprint":false},{"year":2019,"finding":"The Fml1 (FANCM ortholog) interaction with Mhf1-Mhf2 via its C-terminal domain is required for suppression of inter-fork strand annealing (IFSA) during DNA replication termination. Fml1 can catalyze regressed fork restoration in vitro, providing a plausible mechanism for IFSA suppression.","method":"Fission yeast genetics (C-terminal domain mutants, epistasis), in vitro regressed fork restoration assay","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro biochemical assay combined with genetic epistasis, single lab","pmids":["31855181"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of the chicken FANCM (MHF interaction region)-MHF1-MHF2 tripartite complex reveals that FANCM-MHF interaction involves a mixture of hydrophobic/hydrophilic interactions. Under oxidative conditions or in the presence of MPD, FANCM dissociates from MHF while MHF retains its complexed form, indicating the conditional nature of the FANCM-MHF interaction.","method":"X-ray crystallography of tripartite complex, crystallization under various conditions","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure of the complex but single lab, structural characterization without extensive mutagenesis validation","pmids":["33439149"],"is_preprint":false},{"year":2023,"finding":"In fission yeast, Mhf1-Mhf2 (CENP-S/CENP-X counterparts) regulate the spindle assembly checkpoint (SAC). Loss of Mhf2 attenuates the SAC, impairs kinetochore localization of most CCAN components, and alters localization of the Aurora B kinase homolog Ark1 at the kinetochore.","method":"Live-cell microscopy, yeast genetics (deletion mutants), immunofluorescence of SAC components and CCAN proteins","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with multiple phenotypic readouts (SAC, CCAN localization, kinase localization), single lab","pmids":["36537249"],"is_preprint":false},{"year":2025,"finding":"CENP-X is recruited to DNA double-strand breaks in live HeLa cells with a half-time of ~100 s and removed with a half-time of ~2000 s. Recruitment occurs in G1, S, and G2 phases, with delayed but stronger recruitment in G2. CENP-X recruitment occurs simultaneously with CENP-S, immediately after ATM activation and RNF8-RNF168 activity, and removal coincides with RPA loading and RAD51 assembly, placing CENP-X at DSBs during early chromatin remodeling, pathway choice, and resection.","method":"Live-cell microirradiation, quantitative fluorescence microscopy, cell cycle phase analysis, integration with published DDR factor timelines","journal":"DNA repair","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct live-cell imaging with quantitative kinetics and cell-cycle analysis, single lab, no functional validation of the timing","pmids":["40450933"],"is_preprint":false}],"current_model":"CENPX (CENP-X/MHF2) is a histone-fold protein that functions in two distinct contexts: (1) at centromeres, it forms a CENP-S/CENP-X heterodimer and a larger CENP-T-W-S-X nucleosome-like complex that binds linker DNA, induces positive supercoils, is required for outer kinetochore assembly and spindle assembly checkpoint signaling, and assembles de novo in S/G2 phase; (2) in DNA damage repair, it associates with FANCM as MHF2, stimulates FANCM branch migration activity on branched DNA structures (preferring DNA forks/Holliday junctions), is recruited to DSBs in an RSF1- and ATM-dependent manner, and promotes FANCD2/FANCI monoubiquitination and NHEJ via XRCC4 loading, while also suppressing crossovers and chromosomal rearrangements at centromere repeats."},"narrative":{"mechanistic_narrative":"CENPX (CENP-X/MHF2) is a small histone-fold protein that operates at the interface of kinetochore assembly and DNA repair through a single conserved heterodimerization partner, CENP-S (MHF1) [PMID:19620631, PMID:20347429]. At centromeres, CENP-X is a constitutive kinetochore component whose loss reduces outer kinetochore plate size and increases intrakinetochore distance, establishing it as essential for stable outer kinetochore assembly [PMID:19620631]; with CENP-S it integrates into the larger CENP-T-W-S-X complex that preferentially engages ~100 bp of linker DNA, forms a (CENP-T-W-S-X)2 assembly, and induces positive DNA supercoils, with the DNA-binding and supercoiling activity residing in the CENP-T/CENP-W subunits rather than CENP-S/X [PMID:24234442]. CENP-S/X assembles de novo at centromeres during S and G2 phase, with CENP-X exchanging far more rapidly than CENP-S, and the complex is required for spindle assembly checkpoint signaling and for kinetochore loading of CCAN components [PMID:24522885, PMID:36537249]. In its second role, the CENP-S/CENP-X (MHF1/MHF2) heterodimer associates with the FANCM helicase to form a histone-fold tetramer that binds branched DNA—preferring forks and four-way junctions—and stimulates FANCM branch migration activity, with the structural basis defined by crystal structures of the MHF tetramer alone, bound to FANCM, and bound to DNA [PMID:20347429, PMID:22510687, PMID:24390579]. Through this activity CENP-X promotes FANCD2 monoubiquitination and chromatin loading of the Fanconi anemia core complex [PMID:20347429], and is recruited to double-strand breaks in an RSF1- and ATM-dependent manner where it promotes XRCC4 loading and non-homologous end-joining while being dispensable for homologous recombination [PMID:23974106, PMID:24800743]. At centromere DNA repeats it acts with FANCM to suppress crossovers and gross chromosomal rearrangements [PMID:28977643].","teleology":[{"year":2009,"claim":"Established CENP-X as a bona fide constitutive kinetochore protein with a defined structural role, answering whether it contributes to kinetochore architecture.","evidence":"Genetic knockouts in chicken DT40 and siRNA in HeLa cells with live imaging and electron microscopy of the outer plate","pmids":["19620631"],"confidence":"High","gaps":["Did not define the biochemical activity of the CENP-S/X dimer","Mechanism linking CENP-X loss to outer plate shrinkage unresolved"]},{"year":2010,"claim":"Revealed CENP-X has a second identity as MHF2, partnering with MHF1/CENP-S to bind DNA and stimulate FANCM branch migration, connecting it to the Fanconi anemia pathway.","evidence":"Co-IP, protein stability and DNA-binding assays, branch migration assay, siRNA with MMC/CPT sensitivity in cells","pmids":["20347429"],"confidence":"High","gaps":["Structural basis of FANCM engagement not yet defined","DNA substrate preference not yet mapped"]},{"year":2012,"claim":"Defined at atomic resolution how the MHF1-MHF2 tetramer forms and engages FANCM, establishing the architectural plasticity underlying FANCM recruitment.","evidence":"X-ray crystallography of MHF1-MHF2 alone and with FANCM fragment, plus in vivo localization; complemented by yeast structural/genetic analysis of the heterotetramer interface","pmids":["22510687","22325783"],"confidence":"High","gaps":["Did not capture the DNA-bound state","Functional consequences of supercoiling vs branch migration not separated"]},{"year":2013,"claim":"Distinguished the centromeric CENP-T-W-S-X complex's biochemical behavior, showing it binds linker DNA and induces positive supercoils with the activity carried by CENP-T/W rather than CENP-S/X.","evidence":"In vitro DNA-binding and supercoiling assays with mutagenesis and kinetochore targeting assays","pmids":["24234442"],"confidence":"High","gaps":["Specific contribution of CENP-X to complex stability not isolated","Functional role of positive supercoiling in vivo unresolved"]},{"year":2013,"claim":"Placed CENP-S/X within the DSB repair pathway, showing RSF1-dependent assembly that promotes NHEJ via XRCC4 while being dispensable for HR.","evidence":"siRNA, laser microirradiation, NHEJ/HR repair assays; SAXS analysis of the (MHF1-MHF2)4 octamer DNA-binding surface; fission yeast genetics separating repair from centromere functions and demonstrating centromere-repeat crossover suppression","pmids":["23974106","23886707","24026537","28977643"],"confidence":"Medium","gaps":["Single-lab pathway placement for NHEJ","Mechanism by which CENP-S/X directs XRCC4 loading unknown","SAXS surface patch not validated by mutagenesis"]},{"year":2014,"claim":"Defined the upstream recruitment signal and DNA recognition mode for CENP-X at breaks, showing ATM-RSF1-dependent recruitment that drives FANCD2/FANCI monoubiquitination and a structural basis for branched-DNA sensing.","evidence":"Co-IP and laser microirradiation with ubiquitination assays; crystal structure of MHF-DNA complex with biochemical and yeast genetic validation; quantitative live-cell imaging of de novo centromere assembly by FCCS/FRET/FRAP","pmids":["24800743","24390579","24522885"],"confidence":"High","gaps":["How ATM/RSF1 selectivity for chromatin sites is achieved unresolved","Coupling between branched-DNA binding and FA ubiquitination not mechanistically closed"]},{"year":2019,"claim":"Extended MHF-FANCM function to replication termination, showing the interaction suppresses inter-fork strand annealing.","evidence":"Fission yeast C-terminal domain mutants with epistasis and in vitro regressed-fork restoration assay","pmids":["31855181"],"confidence":"Medium","gaps":["Demonstrated in yeast Fml1, human relevance not confirmed","Direct role of CENP-X in fork restoration not isolated"]},{"year":2021,"claim":"Showed the FANCM-MHF interaction is conditional, dissociating under oxidative conditions while the MHF dimer persists, hinting at a regulatable switch.","evidence":"X-ray crystallography of the chicken FANCM-MHF1-MHF2 tripartite complex under varied conditions","pmids":["33439149"],"confidence":"Medium","gaps":["Physiological trigger for dissociation not established","No mutagenesis validation of the conditional interface"]},{"year":2023,"claim":"Connected the centromeric histone-fold pair to spindle assembly checkpoint control and CCAN/Aurora B localization, broadening CENP-X's mitotic role beyond static architecture.","evidence":"Fission yeast deletion mutants with live imaging and immunofluorescence of SAC and CCAN components","pmids":["36537249"],"confidence":"Medium","gaps":["Mechanism linking Mhf2 loss to Ark1 mislocalization unknown","Human CENP-X role in SAC not directly tested"]},{"year":2025,"claim":"Quantified the timing of CENP-X at DSBs, placing it during early chromatin remodeling and pathway choice across all interphase stages.","evidence":"Live-cell microirradiation with quantitative recruitment/removal kinetics and cell-cycle phase analysis","pmids":["40450933"],"confidence":"Medium","gaps":["No functional validation of the observed timing","Whether removal kinetics are causally linked to RPA/RAD51 loading not tested"]},{"year":null,"claim":"How CENP-X is partitioned between its centromeric and DNA-repair pools, and whether the conditional FANCM dissociation regulates this switch in human cells, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No mechanism for routing CENP-S/X between kinetochore and DSB pools","Regulatory triggers for FANCM release not defined in vivo"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1,4,7,10]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,5,6]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,8]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[4,5,6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,8]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,8,14]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,5,6]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[4,8]}],"complexes":["CENP-S/CENP-X (MHF1-MHF2) heterodimer","CENP-T-W-S-X complex","FANCM-MHF complex","kinetochore (CCAN)"],"partners":["CENPS","FANCM","CENPT","CENPW","RSF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"A8MT69","full_name":"Centromere protein X","aliases":["FANCM-associated histone fold protein 2","FANCM-interacting histone fold protein 2","Fanconi anemia-associated polypeptide of 10 kDa","Retinoic acid-inducible gene D9 protein homolog","Stimulated by retinoic acid gene 13 protein homolog"],"length_aa":81,"mass_kda":9.0,"function":"DNA-binding component of the Fanconi anemia (FA) core complex. Required for the normal activation of the FA pathway, leading to monoubiquitination of the FANCI-FANCD2 complex in response to DNA damage, cellular resistance to DNA cross-linking drugs, and prevention of chromosomal breakage (PubMed:20347428, PubMed:20347429). In complex with CENPS (MHF heterodimer), crucial cofactor for FANCM in both binding and ATP-dependent remodeling of DNA. Stabilizes FANCM. In complex with CENPS and FANCM (but not other FANC proteins), rapidly recruited to blocked forks and promotes gene conversion at blocked replication forks (PubMed:20347428, PubMed:20347429). In complex with CENPS, CENPT and CENPW (CENP-T-W-S-X heterotetramer), involved in the formation of a functional kinetochore outer plate, which is essential for kinetochore-microtubule attachment and faithful mitotic progression (PubMed:19620631). As a component of MHF and CENP-T-W-S-X complexes, binds DNA and bends it to form a nucleosome-like structure (PubMed:20347428, PubMed:20347429). DNA-binding function is fulfilled in the presence of CENPS, with the following preference for DNA substates: Holliday junction > double-stranded > splay arm > single-stranded. Does not bind DNA on its own (PubMed:20347429)","subcellular_location":"Nucleus; Chromosome, centromere; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/A8MT69/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CENPX","classification":"Not Classified","n_dependent_lines":276,"n_total_lines":1208,"dependency_fraction":0.22847682119205298},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CENPX","total_profiled":1310},"omim":[{"mim_id":"615128","title":"CENTROMERIC PROTEIN X; CENPX","url":"https://www.omim.org/entry/615128"},{"mim_id":"609130","title":"CENTROMERIC PROTEIN S; CENPS","url":"https://www.omim.org/entry/609130"},{"mim_id":"603386","title":"THYROID CARCINOMA, NONMEDULLARY, WITH OR WITHOUT CELL OXYPHILIA","url":"https://www.omim.org/entry/603386"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CENPX"},"hgnc":{"alias_symbol":["MGC14480","FAAP10","CENP-X"],"prev_symbol":["STRA13","MHF2"]},"alphafold":{"accession":"A8MT69","domains":[{"cath_id":"-","chopping":"45-81","consensus_level":"medium","plddt":98.3362,"start":45,"end":81}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/A8MT69","model_url":"https://alphafold.ebi.ac.uk/files/AF-A8MT69-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-A8MT69-F1-predicted_aligned_error_v6.png","plddt_mean":92.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CENPX","jax_strain_url":"https://www.jax.org/strain/search?query=CENPX"},"sequence":{"accession":"A8MT69","fasta_url":"https://rest.uniprot.org/uniprotkb/A8MT69.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/A8MT69/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/A8MT69"}},"corpus_meta":[{"pmid":"20347429","id":"PMC_20347429","title":"MHF1-MHF2, a histone-fold-containing protein complex, participates in the Fanconi anemia pathway via FANCM.","date":"2010","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/20347429","citation_count":166,"is_preprint":false},{"pmid":"21751032","id":"PMC_21751032","title":"The ABCs of CENPs.","date":"2011","source":"Chromosoma","url":"https://pubmed.ncbi.nlm.nih.gov/21751032","citation_count":164,"is_preprint":false},{"pmid":"19620631","id":"PMC_19620631","title":"The CENP-S complex is essential for the stable assembly of outer kinetochore structure.","date":"2009","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/19620631","citation_count":127,"is_preprint":false},{"pmid":"28673972","id":"PMC_28673972","title":"FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres.","date":"2017","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/28673972","citation_count":125,"is_preprint":false},{"pmid":"25038251","id":"PMC_25038251","title":"FANCM-associated proteins MHF1 and MHF2, but not the other Fanconi anemia factors, limit meiotic crossovers.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25038251","citation_count":84,"is_preprint":false},{"pmid":"24234442","id":"PMC_24234442","title":"The centromeric nucleosome-like CENP-T-W-S-X complex induces positive supercoils into DNA.","date":"2013","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/24234442","citation_count":65,"is_preprint":false},{"pmid":"22510687","id":"PMC_22510687","title":"The structure of the FANCM-MHF complex reveals physical features for functional assembly.","date":"2012","source":"Nature 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Section F, Structural biology communications","url":"https://pubmed.ncbi.nlm.nih.gov/33439149","citation_count":2,"is_preprint":false},{"pmid":"40450933","id":"PMC_40450933","title":"Dynamics of chromatin factors RSF1, CENPS and CENPX at DNA damage sites.","date":"2025","source":"DNA repair","url":"https://pubmed.ncbi.nlm.nih.gov/40450933","citation_count":1,"is_preprint":false},{"pmid":"27193847","id":"PMC_27193847","title":"Biochemical and Structural Analysis of Kinetochore Histone-Fold Complexes.","date":"2016","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/27193847","citation_count":1,"is_preprint":false},{"pmid":"40067534","id":"PMC_40067534","title":"Towards identification of a holocentromere marker in the lepidopteran model Spodoptera frugiperda.","date":"2025","source":"Chromosoma","url":"https://pubmed.ncbi.nlm.nih.gov/40067534","citation_count":0,"is_preprint":false},{"pmid":"38948269","id":"PMC_38948269","title":"[Homozygous Variant of FANCM of the Fanconi Anemia Pathway Causes Premature Ovarian Insufficiency: Investigation of the Pathogenic Mechanism].","date":"2024","source":"Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition","url":"https://pubmed.ncbi.nlm.nih.gov/38948269","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":19760,"output_tokens":4225,"usd":0.061328,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11982,"output_tokens":3736,"usd":0.076655,"stage2_stop_reason":"end_turn"},"total_usd":0.137983,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"CENP-X was identified as a new constitutive kinetochore protein forming a subcomplex with CENP-S. CENP-X-deficient chicken DT40 and human HeLa cells show abnormal mitotic behavior, and kinetochore localization of CENP-X requires CENP-T or CENP-K. Loss of CENP-X results in a significant reduction in outer kinetochore plate size and increased intrakinetochore distance, demonstrating its essential role in stable outer kinetochore assembly.\",\n      \"method\": \"Genetic knockouts in chicken DT40 cells, siRNA knockdown in HeLa cells, live cell imaging, electron microscopy of kinetochore outer plate\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal localization experiments, KO/KD in two cell systems with defined structural phenotype, live imaging\",\n      \"pmids\": [\"19620631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MHF2 (CENP-X) and MHF1 (CENP-S) form a histone-fold-containing heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. Suppression of MHF1 destabilizes FANCM and MHF2, impairs FANCD2 monoubiquitination and foci formation, causes defective chromatin localization of FA core complex proteins, and increases MMC-induced chromosome aberrations.\",\n      \"method\": \"Co-immunoprecipitation, protein stability assays, DNA-binding biochemical assays, branch migration activity assay, siRNA knockdown with cellular phenotyping (immunofluorescence, MMC/CPT sensitivity)\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical reconstitution of DNA-binding and branch migration activity, combined with cellular loss-of-function phenotypes, replicated across multiple assays\",\n      \"pmids\": [\"20347429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of the MHF1-MHF2 (CENP-S/CENP-X) complex reveals that they form a compact tetramer. FANCM binds this tetramer through a 'dual-V' shaped structure, and FANCM-F together with (MHF1-MHF2)2 constitutes a new DNA-binding site coupled to the canonical L1L2 region. Perturbation of MHF-FANCM structural plasticity alters FANCM localization in vivo.\",\n      \"method\": \"X-ray crystallography of MHF1-MHF2 alone and in complex with FANCM fragment, in vivo localization assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure at atomic resolution with functional validation of FANCM localization in vivo\",\n      \"pmids\": [\"22510687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The yeast MHF complex (Mhf1-Mhf2) adopts a histone-fold architecture structurally similar to the (H3-H4)2 heterotetramer. The heterotetrameric assembly is essential for the function of the complex in DNA repair, as shown by genetic data with interface mutants.\",\n      \"method\": \"X-ray crystallography, yeast genetics (mutant analysis of heterotetramer interfaces)\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with functional genetic validation in yeast\",\n      \"pmids\": [\"22325783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The CENP-T-W-S-X complex binds preferentially to ~100 bp of linker DNA (not nucleosome-bound DNA), forms a (CENP-T-W-S-X)2 structure, and induces positive DNA supercoils in contrast to canonical nucleosomes. DNA-binding regions in CENP-T or CENP-W (but not CENP-S or CENP-X) are required for positive supercoiling activity and kinetochore targeting of the complex.\",\n      \"method\": \"DNA-binding assays, DNA supercoiling assay, mutagenesis of DNA-binding regions, kinetochore targeting assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and functional kinetochore targeting assays in a single rigorous study\",\n      \"pmids\": [\"24234442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RSF1 facilitates the assembly of CENP-S and CENP-X at sites of DNA damage. Upon incorporation by RSF1, CENP-S and CENP-X promote assembly of the NHEJ factor XRCC4 at damaged chromatin, thereby promoting non-homologous end-joining. CENP-S and CENP-X are dispensable for homologous recombination.\",\n      \"method\": \"siRNA knockdown, laser microirradiation, immunofluorescence at DSB sites, NHEJ and HR repair assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi loss-of-function with defined DSB repair phenotypes and pathway placement, single lab\",\n      \"pmids\": [\"23974106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RSF1, in an ATM-dependent manner, recruits CENP-X (MHF2) and CENP-S (MHF1) to DSB sites. CENP-X/MHF2 and CENP-S/MHF1 in turn regulate mono-ubiquitination of FANCD2 and FANCI at DSBs. The ATM-RSF1 interaction is dependent on DSBs and ATM kinase activity.\",\n      \"method\": \"Co-immunoprecipitation, laser microirradiation/immunofluorescence, siRNA knockdown, FANCD2/FANCI ubiquitination assays\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, RNAi with pathway-level phenotype, single lab\",\n      \"pmids\": [\"24800743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of human MHF1-MHF2 (CENP-S/CENP-X) bound to DNA reveals that MHF senses branched DNA by engaging two duplex arms simultaneously. Biochemical analyses confirm MHF preferentially binds DNA forks and four-way junctions over dsDNA. Mutations at the observed DNA-binding interface reduce cellular resistance to DNA damage.\",\n      \"method\": \"X-ray crystallography of MHF-DNA complex, DNA-binding biochemical assays, yeast genetic experiments with DNA-binding interface mutants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with orthogonal biochemical validation and functional genetic confirmation\",\n      \"pmids\": [\"24390579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CENP-S and CENP-X assemble de novo at centromeres during S phase and G2, increasing approximately 3-4 fold in abundance. FRET and fluorescence cross-correlation spectroscopy show that CENP-S and CENP-X exist in complex in soluble form and at centromeres. CENPX exchanges ~10 times faster than CENP-S at centromeres (t1/2 ~10 min vs ~120 min). CENP-T was identified as a strong CENP-S binding partner at centromeres by fluorescent two-hybrid and FRET, forming a CENP-S/X/T-containing complex near histone H3 but not CENP-A.\",\n      \"method\": \"Fluorescence cross-correlation spectroscopy, FRET, conditional labeling (EdU pulse-chase), FRAP, fluorescent two-hybrid assay\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal live-cell imaging methods (FRAP, FRET, FCCS) with quantitative functional readouts\",\n      \"pmids\": [\"24522885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In fission yeast, Mhf1 and Mhf2 (CENP-S/CENP-X orthologs) perform two distinct functions: DNA repair/recombination (dependent on interaction with the FANCM ortholog Fml1) and centromere localization for chromosome segregation (independent of Fml1). Together with Fml1, they process sister chromatid junctions to aid chromosome segregation; the Mus81-Eme1 endonuclease acts as a failsafe for unresolved junctions.\",\n      \"method\": \"Fission yeast genetics (deletion mutants, epistasis), chromosome segregation assays, centromere localization experiments\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in model organism demonstrating two distinct functional pathways with defined molecular dependencies\",\n      \"pmids\": [\"24026537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A SAXS study revealed that the (MHF1-MHF2)4 octamer presents a long, positively charged patch on its surface that plays a critical role in double-stranded DNA binding, providing the structural basis for anchoring the MHF-FANCM complex to chromatin.\",\n      \"method\": \"Small angle X-ray scattering (SAXS) in combination with crystallographic data\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural method (SAXS) in solution but single lab without mutagenesis validation of the identified patch\",\n      \"pmids\": [\"23886707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In fission yeast, Mhf1/CENP-S and Mhf2/CENP-X histone-fold proteins together with Fml1/FANCM helicase suppress crossovers between centromere DNA repeats and prevent gross chromosomal rearrangements mediated by centromere repeats during mitosis.\",\n      \"method\": \"Fission yeast genetics (deletion mutants), recombination assays at centromere and non-centromere loci, chromosome rearrangement assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined recombination and chromosomal instability phenotypes at centromeres\",\n      \"pmids\": [\"28977643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The Fml1 (FANCM ortholog) interaction with Mhf1-Mhf2 via its C-terminal domain is required for suppression of inter-fork strand annealing (IFSA) during DNA replication termination. Fml1 can catalyze regressed fork restoration in vitro, providing a plausible mechanism for IFSA suppression.\",\n      \"method\": \"Fission yeast genetics (C-terminal domain mutants, epistasis), in vitro regressed fork restoration assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro biochemical assay combined with genetic epistasis, single lab\",\n      \"pmids\": [\"31855181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of the chicken FANCM (MHF interaction region)-MHF1-MHF2 tripartite complex reveals that FANCM-MHF interaction involves a mixture of hydrophobic/hydrophilic interactions. Under oxidative conditions or in the presence of MPD, FANCM dissociates from MHF while MHF retains its complexed form, indicating the conditional nature of the FANCM-MHF interaction.\",\n      \"method\": \"X-ray crystallography of tripartite complex, crystallization under various conditions\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure of the complex but single lab, structural characterization without extensive mutagenesis validation\",\n      \"pmids\": [\"33439149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In fission yeast, Mhf1-Mhf2 (CENP-S/CENP-X counterparts) regulate the spindle assembly checkpoint (SAC). Loss of Mhf2 attenuates the SAC, impairs kinetochore localization of most CCAN components, and alters localization of the Aurora B kinase homolog Ark1 at the kinetochore.\",\n      \"method\": \"Live-cell microscopy, yeast genetics (deletion mutants), immunofluorescence of SAC components and CCAN proteins\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with multiple phenotypic readouts (SAC, CCAN localization, kinase localization), single lab\",\n      \"pmids\": [\"36537249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CENP-X is recruited to DNA double-strand breaks in live HeLa cells with a half-time of ~100 s and removed with a half-time of ~2000 s. Recruitment occurs in G1, S, and G2 phases, with delayed but stronger recruitment in G2. CENP-X recruitment occurs simultaneously with CENP-S, immediately after ATM activation and RNF8-RNF168 activity, and removal coincides with RPA loading and RAD51 assembly, placing CENP-X at DSBs during early chromatin remodeling, pathway choice, and resection.\",\n      \"method\": \"Live-cell microirradiation, quantitative fluorescence microscopy, cell cycle phase analysis, integration with published DDR factor timelines\",\n      \"journal\": \"DNA repair\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell imaging with quantitative kinetics and cell-cycle analysis, single lab, no functional validation of the timing\",\n      \"pmids\": [\"40450933\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CENPX (CENP-X/MHF2) is a histone-fold protein that functions in two distinct contexts: (1) at centromeres, it forms a CENP-S/CENP-X heterodimer and a larger CENP-T-W-S-X nucleosome-like complex that binds linker DNA, induces positive supercoils, is required for outer kinetochore assembly and spindle assembly checkpoint signaling, and assembles de novo in S/G2 phase; (2) in DNA damage repair, it associates with FANCM as MHF2, stimulates FANCM branch migration activity on branched DNA structures (preferring DNA forks/Holliday junctions), is recruited to DSBs in an RSF1- and ATM-dependent manner, and promotes FANCD2/FANCI monoubiquitination and NHEJ via XRCC4 loading, while also suppressing crossovers and chromosomal rearrangements at centromere repeats.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CENPX (CENP-X/MHF2) is a small histone-fold protein that operates at the interface of kinetochore assembly and DNA repair through a single conserved heterodimerization partner, CENP-S (MHF1) [#0, #1]. At centromeres, CENP-X is a constitutive kinetochore component whose loss reduces outer kinetochore plate size and increases intrakinetochore distance, establishing it as essential for stable outer kinetochore assembly [#0]; with CENP-S it integrates into the larger CENP-T-W-S-X complex that preferentially engages ~100 bp of linker DNA, forms a (CENP-T-W-S-X)2 assembly, and induces positive DNA supercoils, with the DNA-binding and supercoiling activity residing in the CENP-T/CENP-W subunits rather than CENP-S/X [#4]. CENP-S/X assembles de novo at centromeres during S and G2 phase, with CENP-X exchanging far more rapidly than CENP-S, and the complex is required for spindle assembly checkpoint signaling and for kinetochore loading of CCAN components [#8, #14]. In its second role, the CENP-S/CENP-X (MHF1/MHF2) heterodimer associates with the FANCM helicase to form a histone-fold tetramer that binds branched DNA—preferring forks and four-way junctions—and stimulates FANCM branch migration activity, with the structural basis defined by crystal structures of the MHF tetramer alone, bound to FANCM, and bound to DNA [#1, #2, #7]. Through this activity CENP-X promotes FANCD2 monoubiquitination and chromatin loading of the Fanconi anemia core complex [#1], and is recruited to double-strand breaks in an RSF1- and ATM-dependent manner where it promotes XRCC4 loading and non-homologous end-joining while being dispensable for homologous recombination [#5, #6]. At centromere DNA repeats it acts with FANCM to suppress crossovers and gross chromosomal rearrangements [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established CENP-X as a bona fide constitutive kinetochore protein with a defined structural role, answering whether it contributes to kinetochore architecture.\",\n      \"evidence\": \"Genetic knockouts in chicken DT40 and siRNA in HeLa cells with live imaging and electron microscopy of the outer plate\",\n      \"pmids\": [\"19620631\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the biochemical activity of the CENP-S/X dimer\", \"Mechanism linking CENP-X loss to outer plate shrinkage unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed CENP-X has a second identity as MHF2, partnering with MHF1/CENP-S to bind DNA and stimulate FANCM branch migration, connecting it to the Fanconi anemia pathway.\",\n      \"evidence\": \"Co-IP, protein stability and DNA-binding assays, branch migration assay, siRNA with MMC/CPT sensitivity in cells\",\n      \"pmids\": [\"20347429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of FANCM engagement not yet defined\", \"DNA substrate preference not yet mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined at atomic resolution how the MHF1-MHF2 tetramer forms and engages FANCM, establishing the architectural plasticity underlying FANCM recruitment.\",\n      \"evidence\": \"X-ray crystallography of MHF1-MHF2 alone and with FANCM fragment, plus in vivo localization; complemented by yeast structural/genetic analysis of the heterotetramer interface\",\n      \"pmids\": [\"22510687\", \"22325783\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture the DNA-bound state\", \"Functional consequences of supercoiling vs branch migration not separated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Distinguished the centromeric CENP-T-W-S-X complex's biochemical behavior, showing it binds linker DNA and induces positive supercoils with the activity carried by CENP-T/W rather than CENP-S/X.\",\n      \"evidence\": \"In vitro DNA-binding and supercoiling assays with mutagenesis and kinetochore targeting assays\",\n      \"pmids\": [\"24234442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific contribution of CENP-X to complex stability not isolated\", \"Functional role of positive supercoiling in vivo unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed CENP-S/X within the DSB repair pathway, showing RSF1-dependent assembly that promotes NHEJ via XRCC4 while being dispensable for HR.\",\n      \"evidence\": \"siRNA, laser microirradiation, NHEJ/HR repair assays; SAXS analysis of the (MHF1-MHF2)4 octamer DNA-binding surface; fission yeast genetics separating repair from centromere functions and demonstrating centromere-repeat crossover suppression\",\n      \"pmids\": [\"23974106\", \"23886707\", \"24026537\", \"28977643\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab pathway placement for NHEJ\", \"Mechanism by which CENP-S/X directs XRCC4 loading unknown\", \"SAXS surface patch not validated by mutagenesis\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the upstream recruitment signal and DNA recognition mode for CENP-X at breaks, showing ATM-RSF1-dependent recruitment that drives FANCD2/FANCI monoubiquitination and a structural basis for branched-DNA sensing.\",\n      \"evidence\": \"Co-IP and laser microirradiation with ubiquitination assays; crystal structure of MHF-DNA complex with biochemical and yeast genetic validation; quantitative live-cell imaging of de novo centromere assembly by FCCS/FRET/FRAP\",\n      \"pmids\": [\"24800743\", \"24390579\", \"24522885\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ATM/RSF1 selectivity for chromatin sites is achieved unresolved\", \"Coupling between branched-DNA binding and FA ubiquitination not mechanistically closed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended MHF-FANCM function to replication termination, showing the interaction suppresses inter-fork strand annealing.\",\n      \"evidence\": \"Fission yeast C-terminal domain mutants with epistasis and in vitro regressed-fork restoration assay\",\n      \"pmids\": [\"31855181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Demonstrated in yeast Fml1, human relevance not confirmed\", \"Direct role of CENP-X in fork restoration not isolated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed the FANCM-MHF interaction is conditional, dissociating under oxidative conditions while the MHF dimer persists, hinting at a regulatable switch.\",\n      \"evidence\": \"X-ray crystallography of the chicken FANCM-MHF1-MHF2 tripartite complex under varied conditions\",\n      \"pmids\": [\"33439149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological trigger for dissociation not established\", \"No mutagenesis validation of the conditional interface\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected the centromeric histone-fold pair to spindle assembly checkpoint control and CCAN/Aurora B localization, broadening CENP-X's mitotic role beyond static architecture.\",\n      \"evidence\": \"Fission yeast deletion mutants with live imaging and immunofluorescence of SAC and CCAN components\",\n      \"pmids\": [\"36537249\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking Mhf2 loss to Ark1 mislocalization unknown\", \"Human CENP-X role in SAC not directly tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Quantified the timing of CENP-X at DSBs, placing it during early chromatin remodeling and pathway choice across all interphase stages.\",\n      \"evidence\": \"Live-cell microirradiation with quantitative recruitment/removal kinetics and cell-cycle phase analysis\",\n      \"pmids\": [\"40450933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional validation of the observed timing\", \"Whether removal kinetics are causally linked to RPA/RAD51 loading not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CENP-X is partitioned between its centromeric and DNA-repair pools, and whether the conditional FANCM dissociation regulates this switch in human cells, remains unresolved.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mechanism for routing CENP-S/X between kinetochore and DSB pools\", \"Regulatory triggers for FANCM release not defined in vivo\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 4, 7, 10]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [4, 5, 6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 8, 14]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 5, 6]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [4, 8]}\n    ],\n    \"complexes\": [\"CENP-S/CENP-X (MHF1-MHF2) heterodimer\", \"CENP-T-W-S-X complex\", \"FANCM-MHF complex\", \"kinetochore (CCAN)\"],\n    \"partners\": [\"CENPS\", \"FANCM\", \"CENPT\", \"CENPW\", \"RSF1\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}