{"gene":"LRR1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2017,"finding":"CRL2Lrr1 (CUL2 E3 ubiquitin ligase with LRR1 as substrate-recognition subunit) ubiquitylates the MCM7 subunit of the CMG helicase specifically during DNA replication termination in Xenopus egg extracts; in its absence, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large replisome subcomplex including DNA Pol ε is retained on chromatin.","method":"Proteomic screen in Xenopus egg extracts combined with depletion of CRL2Lrr1 and functional readouts of CMG unloading and Mcm7 ubiquitylation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical reconstitution in egg extracts, multiple orthogonal readouts (ubiquitylation assay, chromatin fractionation, proteomics), replicated across independent labs","pmids":["28235849"],"is_preprint":false},{"year":2017,"finding":"CUL-2LRR-1 associates with the replisome and drives ubiquitylation and disassembly of the CMG helicase in both C. elegans embryos and Xenopus egg extracts, acting together with CDC-48 cofactors UFD-1 and NPL-4; chromatin recruitment of CUL2LRR1 is a key regulated step during DNA replication termination, and CUL2 neddylation is required for CMG removal from chromatin.","method":"Genetic inactivation in C. elegans combined with biochemical assays in Xenopus egg extracts; epistasis with CDC-48 cofactors; neddylation inhibition","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — independent replication across two model systems (C. elegans and Xenopus), multiple orthogonal methods, consistent with PMID 28235849","pmids":["28368371"],"is_preprint":false},{"year":2010,"finding":"CRL2(LRR-1) ubiquitin ligase promotes degradation of the Cip/Kip CDK inhibitor CKI-1 in C. elegans germline nuclei to drive G1-phase cell cycle progression, and targets cytoplasmic p21 (CDKN1A) in human cells; loss of human CRL2(LRR1) prevents cytoplasmic p21 degradation, leading to p21-mediated inhibition of the Rho/ROCK/LIMK pathway, activation of the actin-depolymerizing protein cofilin, actin cytoskeleton reorganization, and increased cell motility.","method":"RNAi knockdown in C. elegans and human cells; co-immunoprecipitation; ubiquitylation assays; actin cytoskeleton imaging; cell motility assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitylation assay, genetic epistasis via Rho/ROCK/LIMK pathway, cell motility readout), replicated in two organisms","pmids":["21074724"],"is_preprint":false},{"year":2013,"finding":"CRL2(LRR-1) in C. elegans promotes germ cell proliferation by counteracting the ATL-1 DNA replication checkpoint pathway, participates in the mitotic proliferation/meiotic entry decision, and inhibits early meiotic prophase by targeting the HORMA-domain protein HTP-3 for degradation, thereby preventing loading of synaptonemal complex components onto meiotic chromosomes.","method":"Temperature-sensitive cul-2 mutant analysis; genetic epistasis with ATL-1 checkpoint; Western blot for HTP-3 stability; cytological analysis of meiotic progression","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis and protein stability measurements in a single organism with multiple phenotypic readouts, single lab","pmids":["23555289"],"is_preprint":false},{"year":2021,"finding":"In mouse embryonic stem cells, CUL2LRR1 is required for ubiquitylation of CMG-MCM7 during S-phase and subsequent p97-dependent replisome disassembly; a parallel mitotic pathway for CMG disassembly depends on the TRAIP ubiquitin ligase, establishing that metazoan replisome disassembly is regulated by two conserved ubiquitin ligases.","method":"Auxin-inducible degron depletion of CUL2LRR1 and TRAIP in mouse ES cells; chromatin fractionation; MCM7 ubiquitylation assays; cell cycle analysis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional protein depletion system, multiple orthogonal assays (ubiquitylation, chromatin fractionation), corroborated by independent labs","pmids":["33590678"],"is_preprint":false},{"year":2021,"finding":"In human cells, LRR1 loss prevents CMG helicase unloading from chromatin; chromatin-bound replisome components accumulate throughout S phase, sequestering rate-limiting replisome factors and slowing DNA replication; persistent chromatin-bound CMG in G2 activates ATR-mediated G2/M checkpoint and blocks mitosis; LRR1 is an essential gene for human cell division.","method":"siRNA and CRISPR knockout of LRR1 in human cells; live-cell imaging; DNA replication rate assays; chromatin fractionation; ATR checkpoint activation assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO, fractionation, replication rate, checkpoint activation), mechanistically links LRR1 to rate-limiting replisome recycling and G2/M checkpoint","pmids":["34037657"],"is_preprint":false},{"year":2025,"finding":"USP37 deubiquitylase counteracts CMG helicase ubiquitylation by CUL2LRR1: USP37 binds CDC45 (a CMG subunit) via its Pleckstrin-Homology domain at replication forks, and depletion of CUL2LRR1 suppresses the DNA replication stress sensitivity of USP37 mutants, placing CUL2LRR1 as the ubiquitin writer whose activity is reversed by USP37 to protect ongoing replication forks.","method":"Co-immunoprecipitation of USP37 with CMG; structure-guided mutagenesis of USP37 PH domain; genetic epistasis (CUL2LRR1 depletion suppressing Usp37 mutant phenotypes); sensitivity assays to DNA synthesis inhibitors and ATR inhibitors","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, structure-guided mutagenesis, genetic epistasis, multiple stress conditions tested; peer-reviewed publication","pmids":["40411782"],"is_preprint":false},{"year":2001,"finding":"LRR-1 protein specifically interacts with the cytoplasmic domain of 4-1BB (TNFR superfamily member) as identified by yeast two-hybrid screening; overexpression of LRR-1 suppresses NF-κB activation induced by 4-1BB or TRAF2, and down-regulates JNK1 activity induced by 4-1BB, indicating LRR-1 negatively regulates 4-1BB-mediated signaling cascades.","method":"Yeast two-hybrid screening; overexpression with NF-κB reporter assay; JNK1 kinase activity assay","journal":"Molecules and cells","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid identification and overexpression reporter assays only, single lab, no validation of endogenous interaction or in vivo ubiquitylation context","pmids":["11804328"],"is_preprint":false}],"current_model":"LRR1 is the substrate-recognition subunit of the CRL2(LRR1) E3 ubiquitin ligase complex, which ubiquitylates the MCM7 subunit of the CMG (CDC45-MCM-GINS) replicative helicase during DNA replication termination, thereby initiating p97/CDC-48-dependent replisome disassembly and recycling of replisome components for continued S-phase progression; additionally, in human cells CRL2(LRR1) targets cytoplasmic p21/CKI-1 for degradation to regulate Rho/ROCK/LIMK-mediated actin dynamics and cell motility, while the USP37 deubiquitylase antagonizes CRL2(LRR1)-mediated CMG ubiquitylation at active forks to protect cells from premature replisome disassembly during replication stress."},"narrative":{"mechanistic_narrative":"LRR1 is the substrate-recognition subunit of the CRL2(LRR1) cullin-RING E3 ubiquitin ligase that governs replisome disassembly during DNA replication termination [PMID:28235849, PMID:28368371]. CRL2(LRR1) ubiquitylates the MCM7 subunit of the CMG (CDC45-MCM-GINS) replicative helicase specifically at termination, and this neddylation-dependent ubiquitylation triggers p97/CDC-48-driven (UFD-1/NPL-4-assisted) unloading of CMG and its associated replisome from chromatin [PMID:28235849, PMID:28368371]. This activity is conserved across Xenopus egg extracts, C. elegans, mouse embryonic stem cells and human cells, where a parallel TRAIP-dependent route handles mitotic CMG disassembly [PMID:28368371, PMID:33590678]. In human cells LRR1 is essential for cell division: its loss blocks CMG unloading, causes replisome components to accumulate on chromatin throughout S phase and sequesters rate-limiting replisome factors, slowing replication and provoking ATR-dependent G2/M checkpoint arrest [PMID:34037657]. The ubiquitin mark written by CRL2(LRR1) at active forks is reversed by the USP37 deubiquitylase, which binds the CMG subunit CDC45 through its PH domain to protect ongoing forks from premature disassembly under replication stress [PMID:40411782]. Beyond replication, CRL2(LRR1) promotes cell-cycle progression by targeting Cip/Kip CDK inhibitors for degradation—CKI-1 in the C. elegans germline and cytoplasmic p21 in human cells, the latter linking the ligase to Rho/ROCK/LIMK-cofilin actin remodeling and cell motility [PMID:21074724].","teleology":[{"year":2010,"claim":"Before its replication role was known, LRR1 was established as a substrate-recognition subunit of a CRL2 ligase that degrades CDK inhibitors, answering how it influences cell-cycle progression and, in human cells, actin dynamics.","evidence":"RNAi in C. elegans and human cells with Co-IP, ubiquitylation assays and cell motility/actin imaging","pmids":["21074724"],"confidence":"High","gaps":["Does not address any role in DNA replication or CMG helicase regulation","Mechanism linking cytoplasmic p21 to Rho/ROCK shown by pathway epistasis, not direct biochemistry"]},{"year":2013,"claim":"Extended the CRL2(LRR-1) substrate repertoire in the germline, showing it gates the mitosis-to-meiosis decision by degrading the HORMA protein HTP-3 and counteracting the ATL-1 replication checkpoint.","evidence":"Temperature-sensitive cul-2 mutants, genetic epistasis and HTP-3 stability Westerns in C. elegans","pmids":["23555289"],"confidence":"Medium","gaps":["Single organism and single lab","Direct ubiquitylation of HTP-3 by CRL2(LRR-1) not reconstituted"]},{"year":2017,"claim":"Defined the core conserved function: CRL2(LRR1) ubiquitylates CMG-MCM7 specifically at replication termination to trigger replisome unloading, answering how the replicative helicase is removed from completed DNA.","evidence":"Proteomic screen and depletion in Xenopus egg extracts plus genetic inactivation in C. elegans, with chromatin fractionation, ubiquitylation readouts and CDC-48 cofactor/neddylation epistasis","pmids":["28235849","28368371"],"confidence":"High","gaps":["Did not establish requirement in mammalian cells","Structural basis of MCM7 recognition by LRR1 not resolved"]},{"year":2021,"claim":"Established that the LRR1/CMG-disassembly pathway operates in mammalian cells and is essential, defining two parallel ubiquitin-ligase routes (CRL2(LRR1) in S phase, TRAIP in mitosis) and linking failed unloading to replication slowdown and ATR-dependent G2/M arrest.","evidence":"Auxin-inducible degron and CRISPR/siRNA depletion in mouse ES cells and human cells with chromatin fractionation, MCM7 ubiquitylation, replication-rate and checkpoint assays plus live imaging","pmids":["33590678","34037657"],"confidence":"High","gaps":["Identity of rate-limiting recycled replisome factors not fully enumerated","How chromatin recruitment of CRL2(LRR1) is timed to termination remains undefined"]},{"year":2025,"claim":"Identified the counter-regulator: USP37 deubiquitylates CMG to oppose CRL2(LRR1) at active forks, defining LRR1 as the ubiquitin writer whose reversal protects forks during replication stress.","evidence":"Reciprocal Co-IP of USP37 with CMG, structure-guided PH-domain mutagenesis, and genetic epistasis (CUL2LRR1 depletion suppressing USP37-mutant stress sensitivity) in human cells","pmids":["40411782"],"confidence":"High","gaps":["Direct demonstration that USP37 removes the specific CRL2(LRR1)-deposited MCM7 chain not shown","Spatiotemporal switch favoring writing versus erasing at termination vs. active forks unresolved"]},{"year":null,"claim":"How CRL2(LRR1) discriminates terminated from active forks, and how its chromatin recruitment is restricted to termination, remains the central open mechanistic question.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of LRR1-MCM7 recognition","Determinants of termination-specific recruitment unknown"]}],"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:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0,1,4,5]},{"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":[0,2,4]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[5,6]}],"complexes":["CRL2(LRR1) E3 ubiquitin ligase"],"partners":["CUL2","MCM7","CDC45","USP37","CKI-1","CDKN1A","HTP-3","TNFRSF9"],"other_free_text":[]}},"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":97,"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":90,"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":53,"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":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":7883,"output_tokens":2507,"usd":0.030627,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9577,"output_tokens":2673,"usd":0.057355,"stage2_stop_reason":"end_turn"},"total_usd":0.087982,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"CRL2Lrr1 (CUL2 E3 ubiquitin ligase with LRR1 as substrate-recognition subunit) ubiquitylates the MCM7 subunit of the CMG helicase specifically during DNA replication termination in Xenopus egg extracts; in its absence, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large replisome subcomplex including DNA Pol ε is retained on chromatin.\",\n      \"method\": \"Proteomic screen in Xenopus egg extracts combined with depletion of CRL2Lrr1 and functional readouts of CMG unloading and Mcm7 ubiquitylation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical reconstitution in egg extracts, multiple orthogonal readouts (ubiquitylation assay, chromatin fractionation, proteomics), replicated across independent labs\",\n      \"pmids\": [\"28235849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CUL-2LRR-1 associates with the replisome and drives ubiquitylation and disassembly of the CMG helicase in both C. elegans embryos and Xenopus egg extracts, acting together with CDC-48 cofactors UFD-1 and NPL-4; chromatin recruitment of CUL2LRR1 is a key regulated step during DNA replication termination, and CUL2 neddylation is required for CMG removal from chromatin.\",\n      \"method\": \"Genetic inactivation in C. elegans combined with biochemical assays in Xenopus egg extracts; epistasis with CDC-48 cofactors; neddylation inhibition\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independent replication across two model systems (C. elegans and Xenopus), multiple orthogonal methods, consistent with PMID 28235849\",\n      \"pmids\": [\"28368371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CRL2(LRR-1) ubiquitin ligase promotes degradation of the Cip/Kip CDK inhibitor CKI-1 in C. elegans germline nuclei to drive G1-phase cell cycle progression, and targets cytoplasmic p21 (CDKN1A) in human cells; loss of human CRL2(LRR1) prevents cytoplasmic p21 degradation, leading to p21-mediated inhibition of the Rho/ROCK/LIMK pathway, activation of the actin-depolymerizing protein cofilin, actin cytoskeleton reorganization, and increased cell motility.\",\n      \"method\": \"RNAi knockdown in C. elegans and human cells; co-immunoprecipitation; ubiquitylation assays; actin cytoskeleton imaging; cell motility assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitylation assay, genetic epistasis via Rho/ROCK/LIMK pathway, cell motility readout), replicated in two organisms\",\n      \"pmids\": [\"21074724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CRL2(LRR-1) in C. elegans promotes germ cell proliferation by counteracting the ATL-1 DNA replication checkpoint pathway, participates in the mitotic proliferation/meiotic entry decision, and inhibits early meiotic prophase by targeting the HORMA-domain protein HTP-3 for degradation, thereby preventing loading of synaptonemal complex components onto meiotic chromosomes.\",\n      \"method\": \"Temperature-sensitive cul-2 mutant analysis; genetic epistasis with ATL-1 checkpoint; Western blot for HTP-3 stability; cytological analysis of meiotic progression\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis and protein stability measurements in a single organism with multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"23555289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In mouse embryonic stem cells, CUL2LRR1 is required for ubiquitylation of CMG-MCM7 during S-phase and subsequent p97-dependent replisome disassembly; a parallel mitotic pathway for CMG disassembly depends on the TRAIP ubiquitin ligase, establishing that metazoan replisome disassembly is regulated by two conserved ubiquitin ligases.\",\n      \"method\": \"Auxin-inducible degron depletion of CUL2LRR1 and TRAIP in mouse ES cells; chromatin fractionation; MCM7 ubiquitylation assays; cell cycle analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional protein depletion system, multiple orthogonal assays (ubiquitylation, chromatin fractionation), corroborated by independent labs\",\n      \"pmids\": [\"33590678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In human cells, LRR1 loss prevents CMG helicase unloading from chromatin; chromatin-bound replisome components accumulate throughout S phase, sequestering rate-limiting replisome factors and slowing DNA replication; persistent chromatin-bound CMG in G2 activates ATR-mediated G2/M checkpoint and blocks mitosis; LRR1 is an essential gene for human cell division.\",\n      \"method\": \"siRNA and CRISPR knockout of LRR1 in human cells; live-cell imaging; DNA replication rate assays; chromatin fractionation; ATR checkpoint activation assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO, fractionation, replication rate, checkpoint activation), mechanistically links LRR1 to rate-limiting replisome recycling and G2/M checkpoint\",\n      \"pmids\": [\"34037657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP37 deubiquitylase counteracts CMG helicase ubiquitylation by CUL2LRR1: USP37 binds CDC45 (a CMG subunit) via its Pleckstrin-Homology domain at replication forks, and depletion of CUL2LRR1 suppresses the DNA replication stress sensitivity of USP37 mutants, placing CUL2LRR1 as the ubiquitin writer whose activity is reversed by USP37 to protect ongoing replication forks.\",\n      \"method\": \"Co-immunoprecipitation of USP37 with CMG; structure-guided mutagenesis of USP37 PH domain; genetic epistasis (CUL2LRR1 depletion suppressing Usp37 mutant phenotypes); sensitivity assays to DNA synthesis inhibitors and ATR inhibitors\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, structure-guided mutagenesis, genetic epistasis, multiple stress conditions tested; peer-reviewed publication\",\n      \"pmids\": [\"40411782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"LRR-1 protein specifically interacts with the cytoplasmic domain of 4-1BB (TNFR superfamily member) as identified by yeast two-hybrid screening; overexpression of LRR-1 suppresses NF-κB activation induced by 4-1BB or TRAF2, and down-regulates JNK1 activity induced by 4-1BB, indicating LRR-1 negatively regulates 4-1BB-mediated signaling cascades.\",\n      \"method\": \"Yeast two-hybrid screening; overexpression with NF-κB reporter assay; JNK1 kinase activity assay\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid identification and overexpression reporter assays only, single lab, no validation of endogenous interaction or in vivo ubiquitylation context\",\n      \"pmids\": [\"11804328\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"LRR1 is the substrate-recognition subunit of the CRL2(LRR1) E3 ubiquitin ligase complex, which ubiquitylates the MCM7 subunit of the CMG (CDC45-MCM-GINS) replicative helicase during DNA replication termination, thereby initiating p97/CDC-48-dependent replisome disassembly and recycling of replisome components for continued S-phase progression; additionally, in human cells CRL2(LRR1) targets cytoplasmic p21/CKI-1 for degradation to regulate Rho/ROCK/LIMK-mediated actin dynamics and cell motility, while the USP37 deubiquitylase antagonizes CRL2(LRR1)-mediated CMG ubiquitylation at active forks to protect cells from premature replisome disassembly during replication stress.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LRR1 is the substrate-recognition subunit of the CRL2(LRR1) cullin-RING E3 ubiquitin ligase that governs replisome disassembly during DNA replication termination [#0, #1]. CRL2(LRR1) ubiquitylates the MCM7 subunit of the CMG (CDC45-MCM-GINS) replicative helicase specifically at termination, and this neddylation-dependent ubiquitylation triggers p97/CDC-48-driven (UFD-1/NPL-4-assisted) unloading of CMG and its associated replisome from chromatin [#0, #1]. This activity is conserved across Xenopus egg extracts, C. elegans, mouse embryonic stem cells and human cells, where a parallel TRAIP-dependent route handles mitotic CMG disassembly [#1, #4]. In human cells LRR1 is essential for cell division: its loss blocks CMG unloading, causes replisome components to accumulate on chromatin throughout S phase and sequesters rate-limiting replisome factors, slowing replication and provoking ATR-dependent G2/M checkpoint arrest [#5]. The ubiquitin mark written by CRL2(LRR1) at active forks is reversed by the USP37 deubiquitylase, which binds the CMG subunit CDC45 through its PH domain to protect ongoing forks from premature disassembly under replication stress [#6]. Beyond replication, CRL2(LRR1) promotes cell-cycle progression by targeting Cip/Kip CDK inhibitors for degradation—CKI-1 in the C. elegans germline and cytoplasmic p21 in human cells, the latter linking the ligase to Rho/ROCK/LIMK-cofilin actin remodeling and cell motility [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Before its replication role was known, LRR1 was established as a substrate-recognition subunit of a CRL2 ligase that degrades CDK inhibitors, answering how it influences cell-cycle progression and, in human cells, actin dynamics.\",\n      \"evidence\": \"RNAi in C. elegans and human cells with Co-IP, ubiquitylation assays and cell motility/actin imaging\",\n      \"pmids\": [\"21074724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address any role in DNA replication or CMG helicase regulation\", \"Mechanism linking cytoplasmic p21 to Rho/ROCK shown by pathway epistasis, not direct biochemistry\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the CRL2(LRR-1) substrate repertoire in the germline, showing it gates the mitosis-to-meiosis decision by degrading the HORMA protein HTP-3 and counteracting the ATL-1 replication checkpoint.\",\n      \"evidence\": \"Temperature-sensitive cul-2 mutants, genetic epistasis and HTP-3 stability Westerns in C. elegans\",\n      \"pmids\": [\"23555289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single organism and single lab\", \"Direct ubiquitylation of HTP-3 by CRL2(LRR-1) not reconstituted\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined the core conserved function: CRL2(LRR1) ubiquitylates CMG-MCM7 specifically at replication termination to trigger replisome unloading, answering how the replicative helicase is removed from completed DNA.\",\n      \"evidence\": \"Proteomic screen and depletion in Xenopus egg extracts plus genetic inactivation in C. elegans, with chromatin fractionation, ubiquitylation readouts and CDC-48 cofactor/neddylation epistasis\",\n      \"pmids\": [\"28235849\", \"28368371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish requirement in mammalian cells\", \"Structural basis of MCM7 recognition by LRR1 not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established that the LRR1/CMG-disassembly pathway operates in mammalian cells and is essential, defining two parallel ubiquitin-ligase routes (CRL2(LRR1) in S phase, TRAIP in mitosis) and linking failed unloading to replication slowdown and ATR-dependent G2/M arrest.\",\n      \"evidence\": \"Auxin-inducible degron and CRISPR/siRNA depletion in mouse ES cells and human cells with chromatin fractionation, MCM7 ubiquitylation, replication-rate and checkpoint assays plus live imaging\",\n      \"pmids\": [\"33590678\", \"34037657\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of rate-limiting recycled replisome factors not fully enumerated\", \"How chromatin recruitment of CRL2(LRR1) is timed to termination remains undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified the counter-regulator: USP37 deubiquitylates CMG to oppose CRL2(LRR1) at active forks, defining LRR1 as the ubiquitin writer whose reversal protects forks during replication stress.\",\n      \"evidence\": \"Reciprocal Co-IP of USP37 with CMG, structure-guided PH-domain mutagenesis, and genetic epistasis (CUL2LRR1 depletion suppressing USP37-mutant stress sensitivity) in human cells\",\n      \"pmids\": [\"40411782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration that USP37 removes the specific CRL2(LRR1)-deposited MCM7 chain not shown\", \"Spatiotemporal switch favoring writing versus erasing at termination vs. active forks unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CRL2(LRR1) discriminates terminated from active forks, and how its chromatin recruitment is restricted to termination, remains the central open mechanistic question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of LRR1-MCM7 recognition\", \"Determinants of termination-specific recruitment unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 4]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [0, 1, 4, 5]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 4]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"complexes\": [\"CRL2(LRR1) E3 ubiquitin ligase\"],\n    \"partners\": [\"CUL2\", \"MCM7\", \"CDC45\", \"USP37\", \"CKI-1\", \"CDKN1A\", \"HTP-3\", \"TNFRSF9\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}