{"gene":"C1ORF146","run_date":"2026-06-09T22:02:45","timeline":{"discoveries":[{"year":2008,"finding":"Yeast Spo16 is a ZMM protein required for meiotic crossing-over and synaptonemal complex formation; unlike other ZMM mutants, spo16 mutants retain wild-type crossover interference, indicating Spo16 is specifically required for crossover assurance but not interference. Spo16 physically interacts with the ZMM protein Spo22/Zip4.","method":"Genetic analysis of spo16 mutants (spore viability, crossover frequency, interference measurements), co-immunoprecipitation/interaction studies with Spo22/Zip4","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic and biochemical methods, replicated in context of broader ZMM literature","pmids":["18297071"],"is_preprint":false},{"year":2018,"finding":"Spo16, together with Zip2 and Zip4, forms the ZZS (Zip2-Zip4-Spo16) trimeric complex in yeast. Zip2 and Spo16 share structural similarity to the XPF-ERCC1 nuclease but lack endonuclease activity; instead they preferentially bind branched DNA molecules (recombination intermediates) in vitro. The ZZS complex connects recombination events to the chromosome axis through Zip4 interactions.","method":"Co-immunoprecipitation, in vitro DNA-binding assays with branched DNA substrates, structural similarity analysis, genetic epistasis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical reconstitution of DNA binding, structural analysis, and genetic experiments; replicated by independent studies","pmids":["29440262"],"is_preprint":false},{"year":2019,"finding":"Crystal structure of the Zip2:Spo16 subcomplex reveals a heterodimer structurally related to the XPF:ERCC1 endonuclease complex. Zip2:Spo16 lacks an endonuclease active site but binds specific branched DNA structures present in early meiotic recombination intermediates. Mutations in multiple DNA-binding surfaces (central and HhH domains) on the Zip2:Spo16 heterodimer severely compromise DNA binding and crossover formation in vivo.","method":"X-ray crystallography, in vitro DNA-binding assays, structure-guided mutagenesis, yeast genetics","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro binding assays; multiple orthogonal methods in single rigorous study","pmids":["30566683"],"is_preprint":false},{"year":2019,"finding":"Mammalian SPO16 (C1ORF146) is the ortholog of yeast Spo16; it interacts with SHOC1 (mammalian Zip2 ortholog) and TEX11 (mammalian Zip4 ortholog). SPO16 localizes to recombination nodules and is required for stabilization of SHOC1 and proper localization of other ZMM proteins. Deletion of SPO16 in mice causes meiotic DSBs to be repaired without crossover formation, with less severe effects on synapsis.","method":"Co-immunoprecipitation, immunofluorescence localization, mouse knockout (SPO16 deletion), analysis of DSB repair and crossover formation","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo localization, and clean KO mouse with defined meiotic phenotypes; multiple orthogonal methods","pmids":["30746471"],"is_preprint":false},{"year":2019,"finding":"SCRE (synaptonemal complex reinforcing element; C1ORF146) localizes as discrete foci along the central elements of the synaptonemal complex in mice. Deletion of Scre causes unstable synapsis and SCs, meiosis collapse at the late zygotene stage, and infertility in both sexes. SCRE interacts with synaptonemal complex protein SYCP1 and central element protein SYCE3, functioning to reinforce the integrity of central elements.","method":"Mouse knockout (Scre deletion), immunofluorescence localization on meiotic chromosomes, co-immunoprecipitation with SYCP1 and SYCE3, fertility and histological analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotype, reciprocal Co-IP with two binding partners, direct localization experiments with functional consequence","pmids":["30949703"],"is_preprint":false},{"year":2022,"finding":"M1AP interacts with the mammalian ZZS complex components SHOC1, TEX11, and SPO16. SPO16 is required for M1AP localization to chromosomal axes; M1AP colocalizes with TEX11 and promotes TEX11 recruitment to recombination intermediates. This positions SPO16 as a scaffolding component upstream of M1AP-TEX11 axis recruitment.","method":"Co-immunoprecipitation, immunofluorescence localization, mouse knockout models, analysis of recombination intermediates and crossovers","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP of ZZS components, KO mouse models, direct localization with functional consequence; multiple orthogonal methods","pmids":["36440627"],"is_preprint":false},{"year":1990,"finding":"Yeast SPO16 gene (adjacent to SPO12 on opposite DNA strand) is required for efficient spore formation during meiosis; its transcript is meiosis-specific and partially complementary to SPO12 mRNA, though the complementarity itself was shown to have no functional role in meiosis.","method":"Yeast genetics, Northern blot analysis, insertion mutation disrupting complementarity","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast genetic analysis with defined sporulation phenotype; single lab, foundational identification paper","pmids":["2188099"],"is_preprint":false},{"year":2015,"finding":"In budding yeast, Spo16 (as part of ZMM) is required for Zip3-dependent crossover formation; in a tel1Δ background, ZMM-independent crossovers increase, indicating Spo16/ZMM pathway crossovers are a distinct class. Zip3 foci in tel1Δ cells show normal interference, consistent with Spo16 functioning specifically in the interference-sensitive crossover pathway.","method":"Whole-genome recombination mapping, immunofluorescence (Zip3 foci), double-mutant analysis (tel1Δ zip3Δ)","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — whole-genome mapping plus immunofluorescence, single lab, epistasis confirmed by double mutants","pmids":["26305689"],"is_preprint":false},{"year":2015,"finding":"In budding yeast, K. lactis Zip1-mediated crossovers rely on S. cerevisiae Spo16 (as well as Zip3, Zip4, and Mlh3), demonstrating that Spo16 is required downstream of SC assembly for crossover formation and is part of the pathway that couples recombination intermediates to SC assembly.","method":"Interspecies complementation genetics, spore viability assays, immunofluorescence for SC proteins","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with interspecies complementation, single lab, multiple genetic backgrounds tested","pmids":["26114667"],"is_preprint":false},{"year":2019,"finding":"Rice SPO16 ortholog OsPTD interacts with the Zip2 ortholog OsSHOC1 and with OsHEI10, and is required for the same set of crossover formations as OsSHOC1. Bimolecular fluorescence complementation and yeast-three-hybrid assays showed OsSHOC1, OsPTD, OsHEI10, and OsZIP4 can form various heterotrimeric combinations, conserving the ZZS complex function in plants.","method":"Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), yeast three-hybrid, immunofluorescence localization, plant knockout genetics","journal":"Plant physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple interaction assays (BiFC, Y2H, Y3H) and in vivo localization in rice; single lab but orthogonal methods","pmids":["31266799"],"is_preprint":false},{"year":2023,"finding":"A homozygous splicing variant in SPO16 (c.160+8A>G) produces a non-functional SPO16 protein (verified by minigene splicing assay) and is associated with premature ovarian insufficiency, establishing that SPO16 function (binding branched DNA and recruiting ZMM proteins for crossover formation) is essential for ovarian maintenance in humans.","method":"Whole-exome sequencing, minigene splicing assay, clinical genetic analysis","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — minigene functional validation of splicing defect in single patient; single lab, limited to one variant","pmids":["37270785"],"is_preprint":false},{"year":2024,"finding":"Proximity labeling in yeast demonstrates that Spo16 (as part of the ZZS complex) proximity-labels Ecm11 (SC central element protein) in a Zip4-dependent and Zip1-independent manner, placing ZZS upstream of Ecm11 in SC assembly independently of Zip1.","method":"Proximity labeling (BioID/TurboID), mass spectrometry, yeast genetics","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling with mass spectrometry and genetic controls; single lab, novel method","pmids":["39405359"],"is_preprint":false},{"year":2025,"finding":"In human/mouse meiosis, SHOC1 forms a trimeric ZZS complex with SPO16 and TEX11 to bind recombination intermediates after strand invasion. A missense variant in the XPF-like domain of SHOC1 (p.Q590R) impairs branched-DNA binding and reduces SPO16/TEX11 recruitment to recombination intermediates, abolishing crossover formation. This study establishes that SPO16 depends on SHOC1's XPF-like domain for proper recruitment.","method":"Clinical variant analysis, mouse and human meiocyte immunofluorescence, chromatin immunoprecipitation, 3D chromatin structure analysis, crossover assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo localization and functional assays with patient variant; SPO16 role inferred from SHOC1 complex disruption, single lab","pmids":["42258546"],"is_preprint":false},{"year":2025,"finding":"Men with biallelic loss-of-function variants in ZZS genes (including SPO16) show early meiotic arrest characterized by incorrect synapsis of homologous chromosomes, unrepaired DNA double-strand breaks, and incomplete recombination — distinct from M1AP deficiency phenotype, establishing SPO16 as essential for early meiotic recombination progression in humans.","method":"Clinical genetic screening, testicular biopsy histology and immunofluorescence, comparison of meiotic arrest phenotypes across genotypes","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in-depth human testicular phenotyping with defined molecular markers; multi-center study but limited to histological/cytological readouts","pmids":["40374915"],"is_preprint":false}],"current_model":"SPO16/C1ORF146/SCRE is an evolutionarily conserved meiotic protein that functions in two distinct capacities: (1) as part of the ZZS (Zip2/SHOC1–Zip4/TEX11–Spo16/SPO16) trimeric complex, it forms an XPF-ERCC1-like heterodimer with Zip2/SHOC1 that binds branched DNA recombination intermediates (lacking endonuclease activity) to stabilize and channel them toward interference-sensitive crossover formation, and scaffolds other ZMM proteins at recombination nodules; and (2) in mice, as SCRE, it localizes as discrete foci along the synaptonemal complex central element, where it reinforces SC integrity by interacting with SYCP1 and SYCE3, and its loss causes SC instability and meiotic arrest at the late zygotene stage."},"narrative":{"mechanistic_narrative":"C1ORF146/SPO16 (yeast Spo16; mouse SCRE) is an evolutionarily conserved meiosis-specific protein that promotes interference-sensitive crossover formation as a structural component of the ZZS recombination complex [PMID:18297071, PMID:29440262, PMID:30746471]. Within ZZS, Spo16/SPO16 forms an XPF-ERCC1-like heterodimer with Zip2/SHOC1 that lacks endonuclease activity but binds branched DNA recombination intermediates through central and helix-hairpin-helix DNA-binding surfaces, channeling these intermediates toward crossover resolution [PMID:29440262, PMID:30566683]. Zip4/TEX11 links this DNA-binding module to the chromosome axis, and SPO16 is required to stabilize SHOC1 and to scaffold recruitment of additional ZMM factors, including M1AP-dependent TEX11 loading at recombination intermediates [PMID:29440262, PMID:30746471, PMID:36440627]. Genetically, Spo16 acts specifically in the interference-sensitive (Zip3/ZMM-dependent) crossover pathway and is dispensable for crossover interference itself [PMID:18297071, PMID:26305689]. In mice, the same protein (SCRE) localizes as discrete foci along the synaptonemal complex central element and reinforces SC integrity through interactions with SYCP1 and SYCE3, with loss causing unstable synapsis and meiotic collapse at late zygotene [PMID:30949703]. The ZZS module functions upstream of SC central-element assembly, proximity-labeling Ecm11 in a Zip4-dependent, Zip1-independent manner [PMID:39405359]. In humans, biallelic loss-of-function and splicing variants in SPO16 cause meiotic arrest with defective recombination, underlying premature ovarian insufficiency and male infertility [PMID:37270785, PMID:40374915].","teleology":[{"year":1990,"claim":"Identified the yeast SPO16 gene as a meiosis-specific factor required for sporulation, establishing it as a candidate meiotic gene before any mechanism was known.","evidence":"Yeast genetics and Northern blot analysis of a meiosis-specific transcript with an insertion mutation disrupting SPO12 complementarity","pmids":["2188099"],"confidence":"Medium","gaps":["No molecular function assigned","Antisense complementarity with SPO12 shown to be functionally irrelevant","No protein-level characterization"]},{"year":2008,"claim":"Placed Spo16 in the ZMM crossover pathway and distinguished its role: required for crossover assurance/formation but, unusually among ZMM mutants, dispensable for interference.","evidence":"Genetic analysis of spo16 mutants (spore viability, crossover frequency, interference) plus co-IP with Zip4/Spo22 in budding yeast","pmids":["18297071"],"confidence":"High","gaps":["Biochemical activity of Spo16 unknown","Structural basis of Zip4 interaction undefined","No mammalian counterpart yet identified"]},{"year":2015,"claim":"Refined Spo16's genetic placement, showing it operates in the interference-sensitive, Zip3-dependent crossover class and downstream of SC assembly to couple recombination intermediates to crossover resolution.","evidence":"Whole-genome recombination mapping, Zip3 immunofluorescence, tel1Δ double-mutant epistasis, and interspecies Zip1 complementation in budding yeast","pmids":["26305689","26114667"],"confidence":"Medium","gaps":["Molecular substrate of Spo16 still unknown","Direct biochemical activity not tested"]},{"year":2018,"claim":"Defined the ZZS trimeric complex and showed Zip2-Spo16 resemble the XPF-ERCC1 nuclease yet bind branched DNA without cleaving it, providing the first mechanistic model — a structure-specific DNA-binding scaffold rather than a resolvase.","evidence":"Co-IP, in vitro branched-DNA binding assays, structural similarity analysis, and genetic epistasis in yeast","pmids":["29440262"],"confidence":"High","gaps":["Atomic structure not yet solved","Specific DNA contact residues unmapped","Mammalian conservation untested at this stage"]},{"year":2019,"claim":"Solved the Zip2:Spo16 crystal structure, confirming the XPF-ERCC1-like fold lacking an active site and mapping DNA-binding surfaces whose mutation abolishes crossover formation in vivo.","evidence":"X-ray crystallography, in vitro DNA-binding assays, structure-guided mutagenesis, yeast genetics","pmids":["30566683"],"confidence":"High","gaps":["Structure of the full ZZS trimer absent","Dynamics of intermediate channeling not resolved"]},{"year":2019,"claim":"Established mammalian conservation: SPO16 (C1ORF146) is the Spo16 ortholog, interacts with SHOC1 and TEX11, localizes to recombination nodules, and is required to stabilize SHOC1 and load ZMM proteins, with loss yielding crossover-defective DSB repair.","evidence":"Reciprocal co-IP, immunofluorescence, and SPO16-knockout mouse phenotyping of DSB repair and crossovers; rice OsPTD interaction studies extend conservation to plants","pmids":["30746471","31266799"],"confidence":"High","gaps":["Quantitative DNA-binding of mammalian SPO16 not reconstituted","Order of SHOC1/SPO16/TEX11 assembly incompletely defined"]},{"year":2019,"claim":"Revealed a second function for the same protein (SCRE) at the synaptonemal complex central element, reinforcing SC integrity via SYCP1 and SYCE3, with loss causing SC instability and arrest at late zygotene.","evidence":"Scre-knockout mice, immunofluorescence on meiotic chromosomes, co-IP with SYCP1 and SYCE3, fertility and histology","pmids":["30949703"],"confidence":"High","gaps":["Relationship between the SC-reinforcing role and the ZZS DNA-binding role unresolved","Direct binding interface with SYCP1/SYCE3 not structurally defined"]},{"year":2022,"claim":"Positioned SPO16 as an upstream scaffold for M1AP-TEX11 axis recruitment, showing SPO16 is required for M1AP localization to chromosomal axes and TEX11 loading at intermediates.","evidence":"Reciprocal co-IP of ZZS components, immunofluorescence, and knockout mouse models analyzing recombination intermediates and crossovers","pmids":["36440627"],"confidence":"High","gaps":["Direct vs indirect SPO16-M1AP contact not distinguished","Stoichiometry of the M1AP-ZZS assembly unknown"]},{"year":2024,"claim":"Linked ZZS function to SC central-element assembly, showing Spo16/ZZS proximity-labels Ecm11 in a Zip4-dependent, Zip1-independent manner, placing the complex upstream of central-element protein loading.","evidence":"Proximity labeling (BioID/TurboID) with mass spectrometry and genetic controls in yeast","pmids":["39405359"],"confidence":"Medium","gaps":["Proximity labeling does not prove direct contact","Mechanism coupling ZZS to Ecm11 recruitment unresolved"]},{"year":2025,"claim":"Established human disease relevance: biallelic SPO16 loss-of-function and a SHOC1 XPF-domain variant that impairs SPO16/TEX11 recruitment cause meiotic arrest with defective synapsis, unrepaired DSBs, and abolished crossovers, complementing the earlier premature-ovarian-insufficiency splicing variant.","evidence":"Clinical genetics, minigene splicing assay, testicular biopsy histology/immunofluorescence, meiocyte assays, and crossover analysis with patient variants","pmids":["37270785","40374915","42258546"],"confidence":"Medium","gaps":["SPO16 role in human variants partly inferred from SHOC1 disruption","Single or limited patient cohorts per variant","No structural data on human SPO16"]},{"year":null,"claim":"How a single conserved protein mechanistically reconciles its branched-DNA-binding role within ZZS with its physical reinforcement of the SC central element remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of mammalian SPO16 or the full ZZS trimer","Temporal/spatial switch between recombination-nodule and central-element functions undefined","Quantitative biochemistry of mammalian SPO16 DNA binding not reconstituted"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[3,10,13]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1]}],"complexes":["ZZS complex (Zip2/SHOC1–Zip4/TEX11–Spo16/SPO16)","Zip2:Spo16 (SHOC1:SPO16) heterodimer","synaptonemal complex central element"],"partners":["SHOC1","TEX11","M1AP","SYCP1","SYCE3","ZIP4","ZIP2","ECM11"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5VVC0","full_name":"Protein SPO16 homolog","aliases":["Synaptonemal complex reinforcing element"],"length_aa":180,"mass_kda":20.4,"function":"Plays a key role in reinforcing the integrity of the central element of the synaptonemal complex (SC) thereby stabilizing SC, ensuring progression of meiotic prophase I in male and female germ cells (By similarity). Promotes homologous recombination and crossing-over in meiotic prophase I via its association with SHOC1 (By similarity). Required for the localization of TEX11 and MSH4 to recombination intermediates (By similarity)","subcellular_location":"Chromosome","url":"https://www.uniprot.org/uniprotkb/Q5VVC0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/C1ORF146"},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/C1ORF146","total_profiled":1310},"omim":[{"mim_id":"618968","title":"CHROMOSOME 1 OPEN READING FRAME 146; C1ORF146","url":"https://www.omim.org/entry/618968"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Centriolar satellite","reliability":"Approved"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"retina","ntpm":1.9},{"tissue":"testis","ntpm":7.0}],"url":"https://www.proteinatlas.org/search/C1ORF146"},"hgnc":{"alias_symbol":["SPO16","SCRE"],"prev_symbol":[]},"alphafold":{"accession":"Q5VVC0","domains":[{"cath_id":"3.40.50.10130","chopping":"13-138","consensus_level":"high","plddt":91.4544,"start":13,"end":138},{"cath_id":"1.20.5","chopping":"142-172","consensus_level":"high","plddt":90.2229,"start":142,"end":172}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VVC0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VVC0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VVC0-F1-predicted_aligned_error_v6.png","plddt_mean":86.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=C1ORF146","jax_strain_url":"https://www.jax.org/strain/search?query=C1ORF146"},"sequence":{"accession":"Q5VVC0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5VVC0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5VVC0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VVC0"}},"corpus_meta":[{"pmid":"18297071","id":"PMC_18297071","title":"Crossover 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Spo16 physically interacts with the ZMM protein Spo22/Zip4.\",\n      \"method\": \"Genetic analysis of spo16 mutants (spore viability, crossover frequency, interference measurements), co-immunoprecipitation/interaction studies with Spo22/Zip4\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic and biochemical methods, replicated in context of broader ZMM literature\",\n      \"pmids\": [\"18297071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Spo16, together with Zip2 and Zip4, forms the ZZS (Zip2-Zip4-Spo16) trimeric complex in yeast. Zip2 and Spo16 share structural similarity to the XPF-ERCC1 nuclease but lack endonuclease activity; instead they preferentially bind branched DNA molecules (recombination intermediates) in vitro. The ZZS complex connects recombination events to the chromosome axis through Zip4 interactions.\",\n      \"method\": \"Co-immunoprecipitation, in vitro DNA-binding assays with branched DNA substrates, structural similarity analysis, genetic epistasis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical reconstitution of DNA binding, structural analysis, and genetic experiments; replicated by independent studies\",\n      \"pmids\": [\"29440262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structure of the Zip2:Spo16 subcomplex reveals a heterodimer structurally related to the XPF:ERCC1 endonuclease complex. Zip2:Spo16 lacks an endonuclease active site but binds specific branched DNA structures present in early meiotic recombination intermediates. Mutations in multiple DNA-binding surfaces (central and HhH domains) on the Zip2:Spo16 heterodimer severely compromise DNA binding and crossover formation in vivo.\",\n      \"method\": \"X-ray crystallography, in vitro DNA-binding assays, structure-guided mutagenesis, yeast genetics\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro binding assays; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"30566683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mammalian SPO16 (C1ORF146) is the ortholog of yeast Spo16; it interacts with SHOC1 (mammalian Zip2 ortholog) and TEX11 (mammalian Zip4 ortholog). SPO16 localizes to recombination nodules and is required for stabilization of SHOC1 and proper localization of other ZMM proteins. Deletion of SPO16 in mice causes meiotic DSBs to be repaired without crossover formation, with less severe effects on synapsis.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence localization, mouse knockout (SPO16 deletion), analysis of DSB repair and crossover formation\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo localization, and clean KO mouse with defined meiotic phenotypes; multiple orthogonal methods\",\n      \"pmids\": [\"30746471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SCRE (synaptonemal complex reinforcing element; C1ORF146) localizes as discrete foci along the central elements of the synaptonemal complex in mice. Deletion of Scre causes unstable synapsis and SCs, meiosis collapse at the late zygotene stage, and infertility in both sexes. SCRE interacts with synaptonemal complex protein SYCP1 and central element protein SYCE3, functioning to reinforce the integrity of central elements.\",\n      \"method\": \"Mouse knockout (Scre deletion), immunofluorescence localization on meiotic chromosomes, co-immunoprecipitation with SYCP1 and SYCE3, fertility and histological analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotype, reciprocal Co-IP with two binding partners, direct localization experiments with functional consequence\",\n      \"pmids\": [\"30949703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"M1AP interacts with the mammalian ZZS complex components SHOC1, TEX11, and SPO16. SPO16 is required for M1AP localization to chromosomal axes; M1AP colocalizes with TEX11 and promotes TEX11 recruitment to recombination intermediates. This positions SPO16 as a scaffolding component upstream of M1AP-TEX11 axis recruitment.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence localization, mouse knockout models, analysis of recombination intermediates and crossovers\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP of ZZS components, KO mouse models, direct localization with functional consequence; multiple orthogonal methods\",\n      \"pmids\": [\"36440627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Yeast SPO16 gene (adjacent to SPO12 on opposite DNA strand) is required for efficient spore formation during meiosis; its transcript is meiosis-specific and partially complementary to SPO12 mRNA, though the complementarity itself was shown to have no functional role in meiosis.\",\n      \"method\": \"Yeast genetics, Northern blot analysis, insertion mutation disrupting complementarity\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast genetic analysis with defined sporulation phenotype; single lab, foundational identification paper\",\n      \"pmids\": [\"2188099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In budding yeast, Spo16 (as part of ZMM) is required for Zip3-dependent crossover formation; in a tel1Δ background, ZMM-independent crossovers increase, indicating Spo16/ZMM pathway crossovers are a distinct class. Zip3 foci in tel1Δ cells show normal interference, consistent with Spo16 functioning specifically in the interference-sensitive crossover pathway.\",\n      \"method\": \"Whole-genome recombination mapping, immunofluorescence (Zip3 foci), double-mutant analysis (tel1Δ zip3Δ)\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — whole-genome mapping plus immunofluorescence, single lab, epistasis confirmed by double mutants\",\n      \"pmids\": [\"26305689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In budding yeast, K. lactis Zip1-mediated crossovers rely on S. cerevisiae Spo16 (as well as Zip3, Zip4, and Mlh3), demonstrating that Spo16 is required downstream of SC assembly for crossover formation and is part of the pathway that couples recombination intermediates to SC assembly.\",\n      \"method\": \"Interspecies complementation genetics, spore viability assays, immunofluorescence for SC proteins\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with interspecies complementation, single lab, multiple genetic backgrounds tested\",\n      \"pmids\": [\"26114667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Rice SPO16 ortholog OsPTD interacts with the Zip2 ortholog OsSHOC1 and with OsHEI10, and is required for the same set of crossover formations as OsSHOC1. Bimolecular fluorescence complementation and yeast-three-hybrid assays showed OsSHOC1, OsPTD, OsHEI10, and OsZIP4 can form various heterotrimeric combinations, conserving the ZZS complex function in plants.\",\n      \"method\": \"Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), yeast three-hybrid, immunofluorescence localization, plant knockout genetics\",\n      \"journal\": \"Plant physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple interaction assays (BiFC, Y2H, Y3H) and in vivo localization in rice; single lab but orthogonal methods\",\n      \"pmids\": [\"31266799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A homozygous splicing variant in SPO16 (c.160+8A>G) produces a non-functional SPO16 protein (verified by minigene splicing assay) and is associated with premature ovarian insufficiency, establishing that SPO16 function (binding branched DNA and recruiting ZMM proteins for crossover formation) is essential for ovarian maintenance in humans.\",\n      \"method\": \"Whole-exome sequencing, minigene splicing assay, clinical genetic analysis\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — minigene functional validation of splicing defect in single patient; single lab, limited to one variant\",\n      \"pmids\": [\"37270785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Proximity labeling in yeast demonstrates that Spo16 (as part of the ZZS complex) proximity-labels Ecm11 (SC central element protein) in a Zip4-dependent and Zip1-independent manner, placing ZZS upstream of Ecm11 in SC assembly independently of Zip1.\",\n      \"method\": \"Proximity labeling (BioID/TurboID), mass spectrometry, yeast genetics\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling with mass spectrometry and genetic controls; single lab, novel method\",\n      \"pmids\": [\"39405359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In human/mouse meiosis, SHOC1 forms a trimeric ZZS complex with SPO16 and TEX11 to bind recombination intermediates after strand invasion. A missense variant in the XPF-like domain of SHOC1 (p.Q590R) impairs branched-DNA binding and reduces SPO16/TEX11 recruitment to recombination intermediates, abolishing crossover formation. This study establishes that SPO16 depends on SHOC1's XPF-like domain for proper recruitment.\",\n      \"method\": \"Clinical variant analysis, mouse and human meiocyte immunofluorescence, chromatin immunoprecipitation, 3D chromatin structure analysis, crossover assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo localization and functional assays with patient variant; SPO16 role inferred from SHOC1 complex disruption, single lab\",\n      \"pmids\": [\"42258546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Men with biallelic loss-of-function variants in ZZS genes (including SPO16) show early meiotic arrest characterized by incorrect synapsis of homologous chromosomes, unrepaired DNA double-strand breaks, and incomplete recombination — distinct from M1AP deficiency phenotype, establishing SPO16 as essential for early meiotic recombination progression in humans.\",\n      \"method\": \"Clinical genetic screening, testicular biopsy histology and immunofluorescence, comparison of meiotic arrest phenotypes across genotypes\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in-depth human testicular phenotyping with defined molecular markers; multi-center study but limited to histological/cytological readouts\",\n      \"pmids\": [\"40374915\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPO16/C1ORF146/SCRE is an evolutionarily conserved meiotic protein that functions in two distinct capacities: (1) as part of the ZZS (Zip2/SHOC1–Zip4/TEX11–Spo16/SPO16) trimeric complex, it forms an XPF-ERCC1-like heterodimer with Zip2/SHOC1 that binds branched DNA recombination intermediates (lacking endonuclease activity) to stabilize and channel them toward interference-sensitive crossover formation, and scaffolds other ZMM proteins at recombination nodules; and (2) in mice, as SCRE, it localizes as discrete foci along the synaptonemal complex central element, where it reinforces SC integrity by interacting with SYCP1 and SYCE3, and its loss causes SC instability and meiotic arrest at the late zygotene stage.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"C1ORF146/SPO16 (yeast Spo16; mouse SCRE) is an evolutionarily conserved meiosis-specific protein that promotes interference-sensitive crossover formation as a structural component of the ZZS recombination complex [#0, #1, #3]. Within ZZS, Spo16/SPO16 forms an XPF-ERCC1-like heterodimer with Zip2/SHOC1 that lacks endonuclease activity but binds branched DNA recombination intermediates through central and helix-hairpin-helix DNA-binding surfaces, channeling these intermediates toward crossover resolution [#1, #2]. Zip4/TEX11 links this DNA-binding module to the chromosome axis, and SPO16 is required to stabilize SHOC1 and to scaffold recruitment of additional ZMM factors, including M1AP-dependent TEX11 loading at recombination intermediates [#1, #3, #5]. Genetically, Spo16 acts specifically in the interference-sensitive (Zip3/ZMM-dependent) crossover pathway and is dispensable for crossover interference itself [#0, #7]. In mice, the same protein (SCRE) localizes as discrete foci along the synaptonemal complex central element and reinforces SC integrity through interactions with SYCP1 and SYCE3, with loss causing unstable synapsis and meiotic collapse at late zygotene [#4]. The ZZS module functions upstream of SC central-element assembly, proximity-labeling Ecm11 in a Zip4-dependent, Zip1-independent manner [#11]. In humans, biallelic loss-of-function and splicing variants in SPO16 cause meiotic arrest with defective recombination, underlying premature ovarian insufficiency and male infertility [#10, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Identified the yeast SPO16 gene as a meiosis-specific factor required for sporulation, establishing it as a candidate meiotic gene before any mechanism was known.\",\n      \"evidence\": \"Yeast genetics and Northern blot analysis of a meiosis-specific transcript with an insertion mutation disrupting SPO12 complementarity\",\n      \"pmids\": [\"2188099\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular function assigned\", \"Antisense complementarity with SPO12 shown to be functionally irrelevant\", \"No protein-level characterization\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Placed Spo16 in the ZMM crossover pathway and distinguished its role: required for crossover assurance/formation but, unusually among ZMM mutants, dispensable for interference.\",\n      \"evidence\": \"Genetic analysis of spo16 mutants (spore viability, crossover frequency, interference) plus co-IP with Zip4/Spo22 in budding yeast\",\n      \"pmids\": [\"18297071\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical activity of Spo16 unknown\", \"Structural basis of Zip4 interaction undefined\", \"No mammalian counterpart yet identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Refined Spo16's genetic placement, showing it operates in the interference-sensitive, Zip3-dependent crossover class and downstream of SC assembly to couple recombination intermediates to crossover resolution.\",\n      \"evidence\": \"Whole-genome recombination mapping, Zip3 immunofluorescence, tel1Δ double-mutant epistasis, and interspecies Zip1 complementation in budding yeast\",\n      \"pmids\": [\"26305689\", \"26114667\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrate of Spo16 still unknown\", \"Direct biochemical activity not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the ZZS trimeric complex and showed Zip2-Spo16 resemble the XPF-ERCC1 nuclease yet bind branched DNA without cleaving it, providing the first mechanistic model — a structure-specific DNA-binding scaffold rather than a resolvase.\",\n      \"evidence\": \"Co-IP, in vitro branched-DNA binding assays, structural similarity analysis, and genetic epistasis in yeast\",\n      \"pmids\": [\"29440262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure not yet solved\", \"Specific DNA contact residues unmapped\", \"Mammalian conservation untested at this stage\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Solved the Zip2:Spo16 crystal structure, confirming the XPF-ERCC1-like fold lacking an active site and mapping DNA-binding surfaces whose mutation abolishes crossover formation in vivo.\",\n      \"evidence\": \"X-ray crystallography, in vitro DNA-binding assays, structure-guided mutagenesis, yeast genetics\",\n      \"pmids\": [\"30566683\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full ZZS trimer absent\", \"Dynamics of intermediate channeling not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established mammalian conservation: SPO16 (C1ORF146) is the Spo16 ortholog, interacts with SHOC1 and TEX11, localizes to recombination nodules, and is required to stabilize SHOC1 and load ZMM proteins, with loss yielding crossover-defective DSB repair.\",\n      \"evidence\": \"Reciprocal co-IP, immunofluorescence, and SPO16-knockout mouse phenotyping of DSB repair and crossovers; rice OsPTD interaction studies extend conservation to plants\",\n      \"pmids\": [\"30746471\", \"31266799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative DNA-binding of mammalian SPO16 not reconstituted\", \"Order of SHOC1/SPO16/TEX11 assembly incompletely defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a second function for the same protein (SCRE) at the synaptonemal complex central element, reinforcing SC integrity via SYCP1 and SYCE3, with loss causing SC instability and arrest at late zygotene.\",\n      \"evidence\": \"Scre-knockout mice, immunofluorescence on meiotic chromosomes, co-IP with SYCP1 and SYCE3, fertility and histology\",\n      \"pmids\": [\"30949703\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between the SC-reinforcing role and the ZZS DNA-binding role unresolved\", \"Direct binding interface with SYCP1/SYCE3 not structurally defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Positioned SPO16 as an upstream scaffold for M1AP-TEX11 axis recruitment, showing SPO16 is required for M1AP localization to chromosomal axes and TEX11 loading at intermediates.\",\n      \"evidence\": \"Reciprocal co-IP of ZZS components, immunofluorescence, and knockout mouse models analyzing recombination intermediates and crossovers\",\n      \"pmids\": [\"36440627\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect SPO16-M1AP contact not distinguished\", \"Stoichiometry of the M1AP-ZZS assembly unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked ZZS function to SC central-element assembly, showing Spo16/ZZS proximity-labels Ecm11 in a Zip4-dependent, Zip1-independent manner, placing the complex upstream of central-element protein loading.\",\n      \"evidence\": \"Proximity labeling (BioID/TurboID) with mass spectrometry and genetic controls in yeast\",\n      \"pmids\": [\"39405359\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Proximity labeling does not prove direct contact\", \"Mechanism coupling ZZS to Ecm11 recruitment unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established human disease relevance: biallelic SPO16 loss-of-function and a SHOC1 XPF-domain variant that impairs SPO16/TEX11 recruitment cause meiotic arrest with defective synapsis, unrepaired DSBs, and abolished crossovers, complementing the earlier premature-ovarian-insufficiency splicing variant.\",\n      \"evidence\": \"Clinical genetics, minigene splicing assay, testicular biopsy histology/immunofluorescence, meiocyte assays, and crossover analysis with patient variants\",\n      \"pmids\": [\"37270785\", \"40374915\", \"42258546\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SPO16 role in human variants partly inferred from SHOC1 disruption\", \"Single or limited patient cohorts per variant\", \"No structural data on human SPO16\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single conserved protein mechanistically reconciles its branched-DNA-binding role within ZZS with its physical reinforcement of the SC central element remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of mammalian SPO16 or the full ZZS trimer\", \"Temporal/spatial switch between recombination-nodule and central-element functions undefined\", \"Quantitative biochemistry of mammalian SPO16 DNA binding not reconstituted\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [3, 10, 13]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [\n      \"ZZS complex (Zip2/SHOC1–Zip4/TEX11–Spo16/SPO16)\",\n      \"Zip2:Spo16 (SHOC1:SPO16) heterodimer\",\n      \"synaptonemal complex central element\"\n    ],\n    \"partners\": [\n      \"SHOC1\",\n      \"TEX11\",\n      \"M1AP\",\n      \"SYCP1\",\n      \"SYCE3\",\n      \"Zip4\",\n      \"Zip2\",\n      \"Ecm11\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}