{"gene":"LRR1","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":2017,"finding":"CRL2LRR1 is the E3 ubiquitin ligase responsible for ubiquitylation of the CMG helicase subunit Mcm7 during DNA replication termination in vertebrates. Ubiquitylation of Mcm7 by CRL2LRR1 is required for p97 ATPase-dependent unloading of the CMG helicase (and associated replisome components including DNA Pol ε) from chromatin. CRL2LRR1 binding to chromatin is temporally restricted to replication termination.","method":"Proteomic screen in Xenopus egg extracts; loss-of-function (depletion) with chromatin fractionation and ubiquitylation assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (proteomics, depletion, ubiquitylation assay, chromatin fractionation) in Xenopus system; independently replicated","pmids":["28235849"],"is_preprint":false},{"year":2017,"finding":"CUL-2LRR-1 (C. elegans ortholog) associates with the replisome and drives ubiquitylation and CMG disassembly cooperatively with CDC-48 cofactors UFD-1 and NPL-4. Chromatin recruitment of CUL2LRR1 is a key regulated step during DNA replication termination. CRL2LRR1-independent CMG removal during mitosis requires CDC-48 cofactor UBXN-3 (FAF1 ortholog), and partial inactivation of lrr-1 and ubxn-3 causes synthetic lethality.","method":"C. elegans genetics and Xenopus egg extracts; Co-immunoprecipitation, chromatin fractionation, ubiquitylation assays, epistasis (synthetic lethality)","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, ubiquitylation assays, genetic epistasis in two model systems; independently replicated","pmids":["28368371"],"is_preprint":false},{"year":2010,"finding":"CRL2LRR1 ubiquitin ligase targets the Cip/Kip CDK inhibitor CKI-1 for degradation in C. elegans germline nuclei to promote G1-phase cell cycle progression. In human cells, CRL2LRR1 targets cytoplasmic p21(Cip1/WAF1) for degradation, thereby preventing p21 from inhibiting the Rho/ROCK/LIMK pathway; loss of CRL2LRR1 activates cofilin, reorganizes the actin cytoskeleton, and increases cell motility.","method":"In vivo ubiquitylation assays, co-immunoprecipitation, C. elegans genetics, RNAi knockdown, actin cytoskeleton imaging, cell motility assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (genetics, biochemistry, cell biology) across two organisms; identifies substrates and pathway placement","pmids":["21074724"],"is_preprint":false},{"year":2013,"finding":"CRL2LRR-1 in C. elegans promotes germ cell proliferation by counteracting the ATL-1 (ATR) DNA replication checkpoint pathway, participates in the mitosis-to-meiosis entry decision, and inhibits early meiotic prophase by driving degradation of the HORMA domain protein HTP-3 in mitotic germ cells.","method":"Temperature-sensitive cul-2 allele genetics, epistasis analysis, protein stability assays in C. elegans germline","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with defined molecular substrates (HTP-3) in C. elegans; single lab","pmids":["23555289"],"is_preprint":false},{"year":2021,"finding":"In mammalian (mouse embryonic stem) cells, CUL2LRR1 is required for ubiquitylation of CMG-MCM7 during S-phase, leading to replisome disassembly by p97. A second mitotic pathway of CMG disassembly depends on the TRAIP ubiquitin ligase, establishing that replisome disassembly in metazoa is governed by a conserved pair of ubiquitin ligases (CUL2LRR1 and TRAIP).","method":"Auxin-inducible degron depletion of CUL2LRR1 in mouse ES cells, chromatin fractionation, ubiquitylation assays, cell cycle analysis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — clean conditional KO with ubiquitylation assays and chromatin fractionation; directly establishes mammalian CUL2LRR1 function","pmids":["33590678"],"is_preprint":false},{"year":2021,"finding":"In human cells lacking LRR1, CMG helicases fail to unload from chromatin, causing accumulation of chromatin-bound replisome components throughout S phase. This sequestration of rate-limiting replisome components reduces DNA replication rate progressively through S phase, and persistent chromatin-bound CMG during G2 activates an ATR-mediated G2/M checkpoint. LRR1 is essential for human cell division.","method":"CRISPR knockout of LRR1 in human cells, chromatin fractionation, DNA replication rate measurements (EdU pulse-chase), checkpoint activation assays, cell viability","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple functional readouts (replication rate, checkpoint activation, cell division); mechanistic pathway placement","pmids":["34037657"],"is_preprint":false},{"year":2025,"finding":"The USP37 deubiquitylase binds the CMG helicase (via CDC45) at replication forks and counteracts CMG ubiquitylation by CUL2LRR1, thereby protecting active replisomes from premature disassembly. Depletion of CUL2LRR1 suppresses the replication stress sensitivity of USP37 mutants, placing CUL2LRR1 as the relevant ubiquitin ligase that USP37 opposes during S-phase.","method":"Co-immunoprecipitation, structure-guided mutagenesis of USP37 PH domain, conditional CUL2LRR1 depletion, epistasis with Usp37 mutants, replication stress sensitivity assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 — structure-guided mutagenesis + genetic epistasis + Co-IP; demonstrates direct antagonism between USP37 and CUL2LRR1 on CMG","pmids":["40411782"],"is_preprint":false},{"year":2001,"finding":"Human LRR-1 protein was identified as an interactor of the cytoplasmic domain of the 4-1BB TNF receptor family member by yeast two-hybrid. Overexpression of LRR-1 suppressed 4-1BB- and TRAF2-induced NF-κB activation and down-regulated JNK1 activity induced by 4-1BB, indicating LRR-1 negatively regulates 4-1BB-mediated signaling.","method":"Yeast two-hybrid, overexpression NF-κB reporter assay, JNK1 kinase assay","journal":"Molecules and cells","confidence":"Low","confidence_rationale":"Tier 3 — single yeast two-hybrid and overexpression study; no reciprocal Co-IP or loss-of-function validation","pmids":["11804328"],"is_preprint":false}],"current_model":"CRL2LRR1 is an E3 ubiquitin ligase that ubiquitylates the CMG helicase subunit MCM7 (and CKI-1/p21 in other contexts) to trigger p97/CDC48-dependent replisome disassembly at replication termination in S-phase; in mammalian cells a second TRAIP-dependent pathway removes CMG during mitosis, and the USP37 deubiquitylase antagonizes CUL2LRR1 to protect active replisomes from premature disassembly during replication stress."},"narrative":{"teleology":[{"year":2001,"claim":"An initial yeast two-hybrid screen suggested LRR1 could interact with the 4-1BB receptor cytoplasmic domain and negatively regulate NF-κB and JNK1 signaling, providing the first functional hint for LRR1 but without loss-of-function or endogenous validation.","evidence":"Yeast two-hybrid and overexpression reporter/kinase assays in human cells","pmids":["11804328"],"confidence":"Low","gaps":["No reciprocal Co-IP or endogenous interaction confirmed","No loss-of-function validation","Physiological relevance of 4-1BB interaction unresolved"]},{"year":2010,"claim":"CRL2LRR1 was established as a bona fide E3 ligase targeting the CDK inhibitor CKI-1/p21 for degradation, linking LRR1 to cell cycle control in the C. elegans germline and to cytoplasmic p21-dependent actin cytoskeleton regulation in human cells.","evidence":"In vivo ubiquitylation assays, Co-IP, C. elegans genetics, RNAi, actin/motility assays in human cells","pmids":["21074724"],"confidence":"High","gaps":["Whether p21 targeting is direct or through an adaptor was not structurally resolved","Relative importance of cytoplasmic vs. nuclear p21 degradation unclear"]},{"year":2013,"claim":"CRL2LRR1 was shown to counteract the ATR checkpoint and to degrade the HORMA domain protein HTP-3, expanding its substrate repertoire beyond CDK inhibitors and linking it to the mitosis-to-meiosis transition in C. elegans germ cells.","evidence":"Temperature-sensitive cul-2 allele, genetic epistasis, protein stability assays in C. elegans germline","pmids":["23555289"],"confidence":"Medium","gaps":["HTP-3 degradation not demonstrated biochemically with purified components","Whether HTP-3 targeting is conserved in vertebrates is unknown"]},{"year":2017,"claim":"Two independent studies identified CRL2LRR1 as the E3 ligase that ubiquitylates the CMG helicase subunit MCM7 at replication termination, establishing the central mechanism of p97-dependent replisome disassembly and revealing that CRL2LRR1 chromatin recruitment is temporally restricted to converging forks.","evidence":"Proteomic screens, depletion/add-back, ubiquitylation and chromatin fractionation assays in Xenopus egg extracts; Co-IP, epistasis, and synthetic lethality in C. elegans","pmids":["28235849","28368371"],"confidence":"High","gaps":["Structural basis for LRR1 recognition of MCM7 not determined","Signal that recruits CRL2LRR1 specifically to terminated replisomes not identified"]},{"year":2021,"claim":"CUL2LRR1 was validated as the S-phase CMG disassembly ligase in mammalian cells, while TRAIP was identified as the mitotic backup pathway, and loss of LRR1 was shown to be lethal due to progressive replisome sequestration and ATR checkpoint activation.","evidence":"Auxin-degron depletion in mouse ES cells; CRISPR knockout in human cells with replication rate measurements, checkpoint assays, and cell viability","pmids":["33590678","34037657"],"confidence":"High","gaps":["How CRL2LRR1 and TRAIP are switched between S-phase and mitosis is mechanistically unresolved","Whether LRR1 loss contributes to human disease is unknown"]},{"year":2025,"claim":"USP37 was identified as the deubiquitylase that directly opposes CRL2LRR1-mediated CMG ubiquitylation at active replication forks, revealing a regulatory circuit that protects elongating replisomes from premature disassembly during replication stress.","evidence":"Structure-guided PH domain mutagenesis of USP37, Co-IP with CDC45, genetic epistasis between Usp37 and CUL2LRR1 depletion, replication stress sensitivity assays","pmids":["40411782"],"confidence":"High","gaps":["Whether additional DUBs also protect CMG from CRL2LRR1 is untested","Quantitative balance between USP37 and CRL2LRR1 activity at individual forks not measured"]},{"year":null,"claim":"The molecular signal that triggers CRL2LRR1 recruitment specifically to converging/terminated replisomes — and how this recruitment is prevented at active forks — remains the central unresolved question in the field.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of the LRR1–MCM7 interface exists","Post-translational modifications or conformational changes that license CRL2LRR1 binding at termination are unknown","Whether CRL2LRR1 has additional replication-associated substrates beyond MCM7 in vertebrates is uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,1,4,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0,1,4,5,6]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,3,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,4]}],"complexes":["CRL2LRR1 (CUL2–Elongin B/C–LRR1)"],"partners":["CUL2","MCM7","USP37","CDC48","UFD1","NPL4","CDKN1A"],"other_free_text":[]},"mechanistic_narrative":"LRR1 is a substrate recognition subunit of the CRL2 (CUL2–Elongin B/C) E3 ubiquitin ligase that drives replisome disassembly at replication termination by ubiquitylating the CMG helicase subunit MCM7, enabling p97/CDC-48-dependent extraction of the CMG complex and associated replisome components from chromatin [PMID:28235849, PMID:28368371, PMID:33590678]. CRL2LRR1 is recruited to chromatin specifically at terminating replisomes during S-phase, and its loss causes persistent chromatin-bound CMG, progressive slowing of DNA replication through sequestration of rate-limiting replisome factors, ATR-dependent G2/M checkpoint activation, and lethality in human cells [PMID:34037657]. The deubiquitylase USP37 antagonizes CRL2LRR1 at active forks to protect replisomes from premature disassembly during replication stress, while a TRAIP-dependent mitotic pathway provides a backup mechanism for CMG removal when CRL2LRR1-mediated S-phase unloading fails [PMID:40411782, PMID:33590678]. In C. elegans, CRL2LRR1 additionally targets the CDK inhibitor CKI-1 (p21) for degradation to promote G1/S progression in the germline, and in human cells cytoplasmic p21 degradation by CRL2LRR1 regulates the Rho/ROCK/LIMK–cofilin pathway and actin cytoskeleton dynamics [PMID:21074724]."},"prefetch_data":{"uniprot":{"accession":"Q96L50","full_name":"Leucine-rich repeat protein 1","aliases":["4-1BB-mediated-signaling molecule","4-1BBlrr","LRR-repeat protein 1","LRR-1","Peptidylprolyl isomerase-like 5"],"length_aa":414,"mass_kda":46.7,"function":"Substrate recognition subunit of an ECS (Elongin BC-CUL2/5-SOCS-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:15601820). ECS(LRR1) ubiquitinates MCM7 and promotes CMG replisome disassembly by VCP and chromatin extraction during S-phase (By similarity). May negatively regulate the 4-1BB-mediated signaling cascades which result in the activation of NK-kappaB and JNK1 (PubMed:11804328)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96L50/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/LRR1","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LRR1","total_profiled":1310},"omim":[{"mim_id":"609193","title":"LEUCINE-RICH REPEAT PROTEIN 1; LRR1","url":"https://www.omim.org/entry/609193"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear membrane","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"testis","ntpm":27.2}],"url":"https://www.proteinatlas.org/search/LRR1"},"hgnc":{"alias_symbol":["MGC20689","LRR-1"],"prev_symbol":["PPIL5"]},"alphafold":{"accession":"Q96L50","domains":[{"cath_id":"3.80.10.10","chopping":"181-284","consensus_level":"medium","plddt":96.7402,"start":181,"end":284},{"cath_id":"-","chopping":"315-413","consensus_level":"high","plddt":90.0757,"start":315,"end":413}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96L50","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96L50-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96L50-F1-predicted_aligned_error_v6.png","plddt_mean":88.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LRR1","jax_strain_url":"https://www.jax.org/strain/search?query=LRR1"},"sequence":{"accession":"Q96L50","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96L50.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96L50/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96L50"}},"corpus_meta":[{"pmid":"28235849","id":"PMC_28235849","title":"CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination.","date":"2017","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/28235849","citation_count":96,"is_preprint":false},{"pmid":"28368371","id":"PMC_28368371","title":"CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis.","date":"2017","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/28368371","citation_count":88,"is_preprint":false},{"pmid":"33347703","id":"PMC_33347703","title":"Arabidopsis U-box E3 ubiquitin ligase PUB11 negatively regulates drought tolerance by degrading the receptor-like protein kinases LRR1 and KIN7.","date":"2021","source":"Journal of integrative plant biology","url":"https://pubmed.ncbi.nlm.nih.gov/33347703","citation_count":83,"is_preprint":false},{"pmid":"21074724","id":"PMC_21074724","title":"CRL2(LRR-1) targets a CDK inhibitor for cell cycle control in C. elegans and actin-based motility regulation in human cells.","date":"2010","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/21074724","citation_count":52,"is_preprint":false},{"pmid":"23555289","id":"PMC_23555289","title":"CRL2(LRR-1) E3-ligase regulates proliferation and progression through meiosis in the Caenorhabditis elegans germline.","date":"2013","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23555289","citation_count":35,"is_preprint":false},{"pmid":"33590678","id":"PMC_33590678","title":"CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.","date":"2021","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/33590678","citation_count":32,"is_preprint":false},{"pmid":"11804328","id":"PMC_11804328","title":"A novel leucine-rich repeat protein (LRR-1): potential involvement in 4-1BB-mediated signal transduction.","date":"2001","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/11804328","citation_count":27,"is_preprint":false},{"pmid":"34037657","id":"PMC_34037657","title":"LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components.","date":"2021","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/34037657","citation_count":18,"is_preprint":false},{"pmid":"28534133","id":"PMC_28534133","title":"Silencing of the Rice Gene LRR1 Compromises Rice Xa21 Transcript Accumulation and XA21-Mediated Immunity.","date":"2017","source":"Rice (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/28534133","citation_count":10,"is_preprint":false},{"pmid":"32671118","id":"PMC_32671118","title":"The Protective Effect of Low Dose of Lipopolysaccharide Pretreatment on Endotoxin-Induced Uveitis in Rats Is Associated with Downregulation of CSF-1 and Upregulation of LRR-1.","date":"2020","source":"Journal of immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/32671118","citation_count":5,"is_preprint":false},{"pmid":"40411782","id":"PMC_40411782","title":"USP37 protects mammalian cells during DNA replication stress by counteracting CUL2LRR1 and TRAIP.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/40411782","citation_count":4,"is_preprint":false},{"pmid":"24778939","id":"PMC_24778939","title":"Role of the CRL2(LRR-1) E3 ubiquitin-ligase in the development of the germline in C. elegans.","date":"2013","source":"Worm","url":"https://pubmed.ncbi.nlm.nih.gov/24778939","citation_count":2,"is_preprint":false},{"pmid":"39619177","id":"PMC_39619177","title":"LRR1 involved in the abscisic acid signaling pathway to regulate the early growth and development of Arabidopsis thaliana.","date":"2024","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/39619177","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.09.03.610971","title":"USP37 protects mammalian cells during DNA replication stress by counteracting CUL2<sup>LRR1</sup>and TRAIP","date":"2024-09-03","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.03.610971","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.28.25324850","title":"Whole-Genome Sequencing Reveals Individual and Cohort Level Insights into Chromosome 9p Syndromes","date":"2025-03-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.28.25324850","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8332,"output_tokens":2347,"usd":0.0301},"stage2":{"model":"claude-opus-4-6","input_tokens":5629,"output_tokens":2206,"usd":0.124942},"total_usd":0.155042,"stage1_batch_id":"msgbatch_01Mgbec4vM6c9vXht7JVJzss","stage2_batch_id":"msgbatch_01JkuuH5RB2BdJNsgnhjbC6s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"CRL2LRR1 is the E3 ubiquitin ligase responsible for ubiquitylation of the CMG helicase subunit Mcm7 during DNA replication termination in vertebrates. Ubiquitylation of Mcm7 by CRL2LRR1 is required for p97 ATPase-dependent unloading of the CMG helicase (and associated replisome components including DNA Pol ε) from chromatin. CRL2LRR1 binding to chromatin is temporally restricted to replication termination.\",\n      \"method\": \"Proteomic screen in Xenopus egg extracts; loss-of-function (depletion) with chromatin fractionation and ubiquitylation assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (proteomics, depletion, ubiquitylation assay, chromatin fractionation) in Xenopus system; independently replicated\",\n      \"pmids\": [\"28235849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CUL-2LRR-1 (C. elegans ortholog) associates with the replisome and drives ubiquitylation and CMG disassembly cooperatively with CDC-48 cofactors UFD-1 and NPL-4. Chromatin recruitment of CUL2LRR1 is a key regulated step during DNA replication termination. CRL2LRR1-independent CMG removal during mitosis requires CDC-48 cofactor UBXN-3 (FAF1 ortholog), and partial inactivation of lrr-1 and ubxn-3 causes synthetic lethality.\",\n      \"method\": \"C. elegans genetics and Xenopus egg extracts; Co-immunoprecipitation, chromatin fractionation, ubiquitylation assays, epistasis (synthetic lethality)\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, ubiquitylation assays, genetic epistasis in two model systems; independently replicated\",\n      \"pmids\": [\"28368371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CRL2LRR1 ubiquitin ligase targets the Cip/Kip CDK inhibitor CKI-1 for degradation in C. elegans germline nuclei to promote G1-phase cell cycle progression. In human cells, CRL2LRR1 targets cytoplasmic p21(Cip1/WAF1) for degradation, thereby preventing p21 from inhibiting the Rho/ROCK/LIMK pathway; loss of CRL2LRR1 activates cofilin, reorganizes the actin cytoskeleton, and increases cell motility.\",\n      \"method\": \"In vivo ubiquitylation assays, co-immunoprecipitation, C. elegans genetics, RNAi knockdown, actin cytoskeleton imaging, cell motility assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (genetics, biochemistry, cell biology) across two organisms; identifies substrates and pathway placement\",\n      \"pmids\": [\"21074724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CRL2LRR-1 in C. elegans promotes germ cell proliferation by counteracting the ATL-1 (ATR) DNA replication checkpoint pathway, participates in the mitosis-to-meiosis entry decision, and inhibits early meiotic prophase by driving degradation of the HORMA domain protein HTP-3 in mitotic germ cells.\",\n      \"method\": \"Temperature-sensitive cul-2 allele genetics, epistasis analysis, protein stability assays in C. elegans germline\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with defined molecular substrates (HTP-3) in C. elegans; single lab\",\n      \"pmids\": [\"23555289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In mammalian (mouse embryonic stem) cells, CUL2LRR1 is required for ubiquitylation of CMG-MCM7 during S-phase, leading to replisome disassembly by p97. A second mitotic pathway of CMG disassembly depends on the TRAIP ubiquitin ligase, establishing that replisome disassembly in metazoa is governed by a conserved pair of ubiquitin ligases (CUL2LRR1 and TRAIP).\",\n      \"method\": \"Auxin-inducible degron depletion of CUL2LRR1 in mouse ES cells, chromatin fractionation, ubiquitylation assays, cell cycle analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean conditional KO with ubiquitylation assays and chromatin fractionation; directly establishes mammalian CUL2LRR1 function\",\n      \"pmids\": [\"33590678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In human cells lacking LRR1, CMG helicases fail to unload from chromatin, causing accumulation of chromatin-bound replisome components throughout S phase. This sequestration of rate-limiting replisome components reduces DNA replication rate progressively through S phase, and persistent chromatin-bound CMG during G2 activates an ATR-mediated G2/M checkpoint. LRR1 is essential for human cell division.\",\n      \"method\": \"CRISPR knockout of LRR1 in human cells, chromatin fractionation, DNA replication rate measurements (EdU pulse-chase), checkpoint activation assays, cell viability\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple functional readouts (replication rate, checkpoint activation, cell division); mechanistic pathway placement\",\n      \"pmids\": [\"34037657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The USP37 deubiquitylase binds the CMG helicase (via CDC45) at replication forks and counteracts CMG ubiquitylation by CUL2LRR1, thereby protecting active replisomes from premature disassembly. Depletion of CUL2LRR1 suppresses the replication stress sensitivity of USP37 mutants, placing CUL2LRR1 as the relevant ubiquitin ligase that USP37 opposes during S-phase.\",\n      \"method\": \"Co-immunoprecipitation, structure-guided mutagenesis of USP37 PH domain, conditional CUL2LRR1 depletion, epistasis with Usp37 mutants, replication stress sensitivity assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — structure-guided mutagenesis + genetic epistasis + Co-IP; demonstrates direct antagonism between USP37 and CUL2LRR1 on CMG\",\n      \"pmids\": [\"40411782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Human LRR-1 protein was identified as an interactor of the cytoplasmic domain of the 4-1BB TNF receptor family member by yeast two-hybrid. Overexpression of LRR-1 suppressed 4-1BB- and TRAF2-induced NF-κB activation and down-regulated JNK1 activity induced by 4-1BB, indicating LRR-1 negatively regulates 4-1BB-mediated signaling.\",\n      \"method\": \"Yeast two-hybrid, overexpression NF-κB reporter assay, JNK1 kinase assay\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single yeast two-hybrid and overexpression study; no reciprocal Co-IP or loss-of-function validation\",\n      \"pmids\": [\"11804328\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CRL2LRR1 is an E3 ubiquitin ligase that ubiquitylates the CMG helicase subunit MCM7 (and CKI-1/p21 in other contexts) to trigger p97/CDC48-dependent replisome disassembly at replication termination in S-phase; in mammalian cells a second TRAIP-dependent pathway removes CMG during mitosis, and the USP37 deubiquitylase antagonizes CUL2LRR1 to protect active replisomes from premature disassembly during replication stress.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"LRR1 is a substrate recognition subunit of the CRL2 (CUL2–Elongin B/C) E3 ubiquitin ligase that drives replisome disassembly at replication termination by ubiquitylating the CMG helicase subunit MCM7, enabling p97/CDC-48-dependent extraction of the CMG complex and associated replisome components from chromatin [PMID:28235849, PMID:28368371, PMID:33590678]. CRL2LRR1 is recruited to chromatin specifically at terminating replisomes during S-phase, and its loss causes persistent chromatin-bound CMG, progressive slowing of DNA replication through sequestration of rate-limiting replisome factors, ATR-dependent G2/M checkpoint activation, and lethality in human cells [PMID:34037657]. The deubiquitylase USP37 antagonizes CRL2LRR1 at active forks to protect replisomes from premature disassembly during replication stress, while a TRAIP-dependent mitotic pathway provides a backup mechanism for CMG removal when CRL2LRR1-mediated S-phase unloading fails [PMID:40411782, PMID:33590678]. In C. elegans, CRL2LRR1 additionally targets the CDK inhibitor CKI-1 (p21) for degradation to promote G1/S progression in the germline, and in human cells cytoplasmic p21 degradation by CRL2LRR1 regulates the Rho/ROCK/LIMK–cofilin pathway and actin cytoskeleton dynamics [PMID:21074724].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"An initial yeast two-hybrid screen suggested LRR1 could interact with the 4-1BB receptor cytoplasmic domain and negatively regulate NF-κB and JNK1 signaling, providing the first functional hint for LRR1 but without loss-of-function or endogenous validation.\",\n      \"evidence\": \"Yeast two-hybrid and overexpression reporter/kinase assays in human cells\",\n      \"pmids\": [\"11804328\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No reciprocal Co-IP or endogenous interaction confirmed\", \"No loss-of-function validation\", \"Physiological relevance of 4-1BB interaction unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"CRL2LRR1 was established as a bona fide E3 ligase targeting the CDK inhibitor CKI-1/p21 for degradation, linking LRR1 to cell cycle control in the C. elegans germline and to cytoplasmic p21-dependent actin cytoskeleton regulation in human cells.\",\n      \"evidence\": \"In vivo ubiquitylation assays, Co-IP, C. elegans genetics, RNAi, actin/motility assays in human cells\",\n      \"pmids\": [\"21074724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p21 targeting is direct or through an adaptor was not structurally resolved\", \"Relative importance of cytoplasmic vs. nuclear p21 degradation unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"CRL2LRR1 was shown to counteract the ATR checkpoint and to degrade the HORMA domain protein HTP-3, expanding its substrate repertoire beyond CDK inhibitors and linking it to the mitosis-to-meiosis transition in C. elegans germ cells.\",\n      \"evidence\": \"Temperature-sensitive cul-2 allele, genetic epistasis, protein stability assays in C. elegans germline\",\n      \"pmids\": [\"23555289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"HTP-3 degradation not demonstrated biochemically with purified components\", \"Whether HTP-3 targeting is conserved in vertebrates is unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Two independent studies identified CRL2LRR1 as the E3 ligase that ubiquitylates the CMG helicase subunit MCM7 at replication termination, establishing the central mechanism of p97-dependent replisome disassembly and revealing that CRL2LRR1 chromatin recruitment is temporally restricted to converging forks.\",\n      \"evidence\": \"Proteomic screens, depletion/add-back, ubiquitylation and chromatin fractionation assays in Xenopus egg extracts; Co-IP, epistasis, and synthetic lethality in C. elegans\",\n      \"pmids\": [\"28235849\", \"28368371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for LRR1 recognition of MCM7 not determined\", \"Signal that recruits CRL2LRR1 specifically to terminated replisomes not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"CUL2LRR1 was validated as the S-phase CMG disassembly ligase in mammalian cells, while TRAIP was identified as the mitotic backup pathway, and loss of LRR1 was shown to be lethal due to progressive replisome sequestration and ATR checkpoint activation.\",\n      \"evidence\": \"Auxin-degron depletion in mouse ES cells; CRISPR knockout in human cells with replication rate measurements, checkpoint assays, and cell viability\",\n      \"pmids\": [\"33590678\", \"34037657\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CRL2LRR1 and TRAIP are switched between S-phase and mitosis is mechanistically unresolved\", \"Whether LRR1 loss contributes to human disease is unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"USP37 was identified as the deubiquitylase that directly opposes CRL2LRR1-mediated CMG ubiquitylation at active replication forks, revealing a regulatory circuit that protects elongating replisomes from premature disassembly during replication stress.\",\n      \"evidence\": \"Structure-guided PH domain mutagenesis of USP37, Co-IP with CDC45, genetic epistasis between Usp37 and CUL2LRR1 depletion, replication stress sensitivity assays\",\n      \"pmids\": [\"40411782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether additional DUBs also protect CMG from CRL2LRR1 is untested\", \"Quantitative balance between USP37 and CRL2LRR1 activity at individual forks not measured\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular signal that triggers CRL2LRR1 recruitment specifically to converging/terminated replisomes — and how this recruitment is prevented at active forks — remains the central unresolved question in the field.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of the LRR1–MCM7 interface exists\", \"Post-translational modifications or conformational changes that license CRL2LRR1 binding at termination are unknown\", \"Whether CRL2LRR1 has additional replication-associated substrates beyond MCM7 in vertebrates is uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 1, 4, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [0, 1, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 4]}\n    ],\n    \"complexes\": [\n      \"CRL2LRR1 (CUL2–Elongin B/C–LRR1)\"\n    ],\n    \"partners\": [\n      \"CUL2\",\n      \"MCM7\",\n      \"USP37\",\n      \"CDC48\",\n      \"UFD1\",\n      \"NPL4\",\n      \"CDKN1A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}