{"gene":"TICRR","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2010,"finding":"Treslin (TICRR) associates with TopBP1 in Xenopus egg extracts and human cells, and is required for loading of Cdc45 onto chromatin. Depletion of Treslin strongly inhibits chromosomal DNA replication. Treslin binds chromatin independently of TopBP1, but Treslin-TopBP1 association requires Cdk2 activity and is necessary for Cdc45 loading.","method":"Immunodepletion from Xenopus egg extracts, co-immunoprecipitation, siRNA knockdown in human cells, chromatin fractionation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, depletion/add-back in egg extracts, corroborated by human cell knockdown, multiple orthogonal methods in one study","pmids":["20116089"],"is_preprint":false},{"year":2010,"finding":"TICRR (ticrr) associates with TopBP1 via BRCT motifs essential for TopBP1's replication and checkpoint functions, and ticrr deficiency disrupts chromatin binding of pre-initiation complex (pre-IC) components without affecting pre-replication complex (pre-RC) loading, placing TICRR at the pre-IC assembly step.","method":"Zebrafish genetic screen, co-immunoprecipitation, chromatin fractionation, morpholino knockdown","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic screen plus biochemical epistasis (pre-RC vs pre-IC components), replicated in zebrafish and human cells","pmids":["20080954"],"is_preprint":false},{"year":2011,"finding":"Treslin/TICRR contains two conserved CDK phosphorylation sites (orthologous to yeast Sld3 CDK sites) that are essential for DNA replication. Phosphorylation of these sites mediates interaction with the N-terminal pair of BRCT repeats in TopBP1 (ortholog of yeast Dpb11). Replication stress prevents this interaction via the Chk1 checkpoint kinase.","method":"Mutagenesis of CDK sites, co-immunoprecipitation, siRNA knockdown with replication assays, sequence conservation analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis of key CDK sites combined with Co-IP, replication assays, and checkpoint epistasis; independently supported by multiple labs","pmids":["21700459"],"is_preprint":false},{"year":2011,"finding":"Treslin is phosphorylated by Cdk2-cyclin E at a conserved CDK consensus site (S976 in Xenopus, S1000 in humans). This phosphorylation is required for effective association with TopBP1 and for DNA replication. A non-phosphorylatable mutant shows severely diminished TopBP1 binding and replication deficiency.","method":"In vitro kinase assay with recombinant Cdk2-cyclin E, site-directed mutagenesis, co-immunoprecipitation, human cell replication assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus functional rescue, replicated in both Xenopus and human cells","pmids":["21646402"],"is_preprint":false},{"year":2014,"finding":"Chk1 associates specifically with a C-terminal domain of Treslin (TRCT domain). This interaction enables Chk1-catalyzed phosphorylation of Treslin. Abolishing the Treslin-Chk1 interaction results in elevated replication initiation during an unperturbed S phase due to enhanced Cdc45 loading, revealing a Chk1 role in suppressing origin firing during normal S phase.","method":"Co-immunoprecipitation, TRCT domain mutagenesis, EdU incorporation assays, Cdc45 chromatin loading analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mutagenesis abolishing binding combined with functional readout (Cdc45 loading, origin firing), multiple orthogonal methods","pmids":["25557548"],"is_preprint":false},{"year":2013,"finding":"Treslin stimulates ATR-mediated phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner, and the phosphorylation state of Treslin at Ser-1000 is important for this checkpoint activity, indicating Treslin is a dual replication/checkpoint protein.","method":"In vitro ATR kinase assay, cell-based checkpoint assays, phosphorylation site mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Weak — in vitro kinase assay and mutagenesis from single lab, single study","pmids":["23696651"],"is_preprint":false},{"year":2015,"finding":"Overexpression of a phosphomimetic TICRR mutant (TICRR-TESE, mimicking CDK phosphorylation at two key residues) shortens S phase by increasing replication initiation. This effect requires the TICRR region necessary for interaction with MTBP (Mdm2-binding protein), demonstrating that phosphorylated TICRR is limiting for S-phase progression.","method":"Phosphomimetic mutant overexpression, EdU incorporation, DNA fiber analysis, replication cluster analysis in human cells","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphomimetic mutant with multiple replication readouts and domain requirement, single lab with orthogonal methods","pmids":["25737283"],"is_preprint":false},{"year":2017,"finding":"MTBP (Mdm2-binding protein) is a binding partner of Treslin. Depletion of MTBP from Xenopus egg extracts co-depletes Treslin and abolishes DNA replication, which can only be rescued by recombinant Treslin-MTBP together, not either protein alone. MTBP contains a C-terminal domain (CTM) that binds double-stranded and G-quadruplex DNA, and CTM mutants are defective for chromatin localization and Cdc45 loading.","method":"Immunodepletion and add-back in Xenopus egg extracts, DNA-binding assays, chromatin fractionation, human cell S-phase assays, domain mutagenesis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reconstitution-like add-back experiment, mutagenesis, DNA-binding assay, functional validation in Xenopus and human cells","pmids":["28877985"],"is_preprint":false},{"year":2017,"finding":"Ensa (a substrate of Greatwall kinase) controls Treslin protein levels via the ubiquitin-proteasome pathway. Ensa knockdown reduces Treslin levels and extends S phase; this phenotype is rescued by Treslin overexpression. The Gwl/Ensa/PP2A-B55 pathway thus regulates S-phase duration by controlling Treslin degradation.","method":"siRNA knockdown, proteasome inhibitor treatment, rescue experiments with Treslin overexpression, flow cytometry","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — knockdown plus rescue with functional readout, single lab, two orthogonal methods","pmids":["28785014"],"is_preprint":false},{"year":2017,"finding":"Mutant p53 (contact-type, e.g. R273H) enhances the interaction between TopBP1 and Treslin and promotes DNA replication even when Cdk2 is inhibited, bypassing the normal CDK requirement for Treslin-TopBP1 interaction.","method":"Co-immunoprecipitation, BrdU incorporation assays, CDK2 inhibitor treatment, expression of specific p53 mutants","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP plus functional replication assay, single lab with two orthogonal methods","pmids":["28439015"],"is_preprint":false},{"year":2021,"finding":"MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Treslin-MTBP is rate-limiting for replication initiation. DDK activity both increases and strengthens the interaction of Treslin-MTBP with licensed chromatin, and cooperates with CDK to drive Treslin-MTBP interaction with TopBP1 at the pre-IC formation step.","method":"Immunodepletion and add-back in Xenopus egg extracts, biochemical characterization of complex stoichiometry, kinase inhibitor treatments","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — depletion/add-back plus kinase inhibitor experiments in Xenopus system, single lab","pmids":["34699733"],"is_preprint":false},{"year":2021,"finding":"TICRR/TRESLIN protein levels are high in G1 and decrease upon S-phase entry via CRL4-DTL E3 ubiquitin ligase-dependent proteasomal degradation. This mechanism moderates TICRR levels to control the number of active origins during S phase.","method":"Cell cycle synchronization, immunoblotting, siRNA screens targeting E3 ligase components, proteasome inhibitor treatment","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — targeted siRNA screen identifying CRL4-DTL, proteasome inhibitor validation, cell cycle fractionation; single lab","pmids":["34534348"],"is_preprint":false},{"year":2020,"finding":"MTBP (and hence the Treslin-MTBP complex) binds to at least 30,000 sites in the human genome, preferentially at regions of open chromatin containing transcriptional-regulatory elements (promoters, enhancers, super-enhancers) with nucleosome-free DNA (G-quadruplex or AP-1 motifs) and H3K4me2-marked nucleosomes.","method":"ChIP-seq genome-wide mapping of MTBP in human cells, correlation with chromatin accessibility and histone modification datasets","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — genome-wide ChIP-seq localization with functional context, single lab","pmids":["32966791"],"is_preprint":false},{"year":2022,"finding":"TRESLIN-MTBP acts transiently at pre-replication complexes to initiate origin firing and is released after CDC45 recruitment. The dynamic behavior of TRESLIN-MTBP implements a monitoring system that senses the rate of origin firing and the decline of active origins in late S phase, preventing premature entry into G2 independently of ATR/CHK1 kinases.","method":"Live-cell imaging, chromatin fractionation, cell cycle analysis, ATR/CHK1 inhibitor treatments, knockdown experiments","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays with kinase inhibitors and knockdowns, single lab with orthogonal methods","pmids":["36049481"],"is_preprint":false},{"year":2022,"finding":"Chk1 regulates the Treslin-TopBP1 interaction through multiple mechanisms during replication stress: (1) Chk1-dependent decrease in CDK activity reduces phosphorylation of Treslin T968 (but not S1000), and (2) Chk1 directly phosphorylates additional sites on Treslin including S1114, both contributing to dissociation of TopBP1 from Treslin and suppression of origin firing.","method":"Phospho-specific antibodies, CDK inhibitor and Chk1 inhibitor treatments, mutagenesis, replication origin firing assays in cancer cell lines","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-site specific analysis plus mutagenesis plus functional origin firing assays, single lab with multiple orthogonal methods","pmids":["35231445"],"is_preprint":false},{"year":2018,"finding":"DUE-B interacts with Treslin in HeLa cells in a cell-cycle-regulated manner (peaking as cells exit G1). The conserved C-terminal domain of DUE-B is required for its binding to TopBP1, Treslin, Cdc45, and MCM2-7, and for efficient chromatin loading of Treslin, Cdc45, and TopBP1. The DUE-B-Treslin interaction is required for Cdc45 loading onto chromatin.","method":"Co-immunoprecipitation, domain mutagenesis, chromatin fractionation, cell cycle synchronization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus domain mutagenesis plus functional chromatin loading assay, single lab","pmids":["30037903"],"is_preprint":false},{"year":2017,"finding":"CKS proteins (Cks1, Cks2) enhance the ability of Cdk2 to phosphorylate Treslin in vitro, promoting replication checkpoint recovery. CKS binding-defective mutants retain the ability to stimulate Treslin phosphorylation. Silencing of Cks1/Cks2 decreases Treslin phosphorylation in vivo, and CKS overexpression prevents checkpoint-dependent dephosphorylation of Treslin.","method":"In vitro kinase assay, siRNA knockdown, CKS mutant overexpression, phospho-Treslin immunoblotting","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay plus in vivo phosphorylation analysis, single lab with orthogonal in vitro and in vivo methods","pmids":["28739856"],"is_preprint":false},{"year":2025,"finding":"TRESLIN-MTBP is a key limiting firing factor for replication initiation in human cells. Its loading onto phosphorylated MCM2-7 double hexamer (MCM-DH) is controlled by opposing phosphorylation events on MCM-DH by DDK (promoting TRESLIN-MTBP loading) and RIF1-Protein Phosphatase 1 (opposing loading), which determines initiation zones and establishes replication timing.","method":"Genome-wide mapping of TRESLIN-MTBP and MCM, kinase/phosphatase inhibitor treatments, chromatin fractionation in human cells","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide functional mapping plus kinase/phosphatase epistasis, single lab with multiple orthogonal methods","pmids":["41331242"],"is_preprint":false},{"year":2025,"finding":"During G1, MTBP depends on TRESLIN for proper chromatin association, but not during S phase, suggesting two distinct modes of TRESLIN-MTBP chromatin binding. Neither TRESLIN nor MTBP binding to chromatin during G1 requires licensed MCM origins.","method":"Cut&Run genomic binding mapping, cell cycle synchronization, Geminin-based MCM depletion, siRNA knockdown of TRESLIN/MTBP","journal":"Genome biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Cut&Run with cell cycle synchronization and MCM depletion controls, single lab with multiple conditions","pmids":["40624716"],"is_preprint":false},{"year":2022,"finding":"Sequence analysis and structural homology modeling revealed that Treslin/TICRR contains a conserved Ku70-homologous β-barrel fold in its middle domain (M domain), and a von Willebrand factor type A domain in the CIT region. The M domain, Sld3-Treslin domain, and TopBP1/Dpb11 interaction domain together constitute the Sld3-homologous core. Domain mutants expressed in human cells indicate all three Sld3-core domains plus non-conserved terminal domains are required for proper origin firing.","method":"Protein sequence analysis, structural homology modeling, domain mutant expression in human cells with replication assays","journal":"Life science alliance","confidence":"Low","confidence_rationale":"Tier 3-4 / Moderate — domain mutagenesis in human cells provides functional validation, but structural assignment is computational; single lab","pmids":["35091422"],"is_preprint":false},{"year":2025,"finding":"WEE1 kinase inhibition during S phase increases CDK activity, which blocks PCNA-dependent proteasomal degradation of TRESLIN and enhances chromatin association of TRESLIN and MTBP, leading to elevated helicase recruitment and dormant origin firing. A conserved sequence within TRESLIN is required for this CDK-sensitive degradation.","method":"WEE1 inhibitor treatment, CDK inhibitor reversal, PCNA dependency assays, chromatin fractionation, DNA fiber analysis in human cells, domain mutagenesis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological epistasis plus domain mutagenesis plus functional replication readouts; preprint, single lab","pmids":["bio_10.1101_2025.06.10.657920"],"is_preprint":true}],"current_model":"TICRR/TRESLIN is an essential metazoan replication initiation factor (ortholog of yeast Sld3) that forms a stable complex with MTBP (ortholog of Sld7), is phosphorylated by CDK2 (at T968/S1000 in humans), and in its phosphorylated form interacts with TopBP1 BRCT repeats to mediate loading of CDC45 onto the MCM2-7 helicase and assemble the active CMG helicase at replication origins; this central interaction is negatively regulated by Chk1 (which directly phosphorylates Treslin and promotes dephosphorylation of its CDK sites during replication stress) and is subject to CRL4-DTL-mediated proteasomal degradation during S phase, while DDK activity promotes TRESLIN-MTBP loading onto phosphorylated MCM double hexamers and cooperates with CDK to drive pre-initiation complex formation, making TRESLIN-MTBP a key rate-limiting determinant of origin selection, replication timing, and the S/G2 transition."},"narrative":{"mechanistic_narrative":"TICRR/TRESLIN is an essential metazoan DNA replication initiation factor that acts at the pre-initiation complex (pre-IC) assembly step to load CDC45 onto licensed origins, and its depletion abolishes chromosomal DNA replication [PMID:20116089, PMID:20080954]. TRESLIN binds chromatin independently of TopBP1, but its association with the N-terminal BRCT repeats of TopBP1 requires CDK2-cyclin E phosphorylation at conserved CDK consensus sites (S1000/T968 in humans), and this phospho-dependent TRESLIN–TopBP1 interaction is the rate-limiting event for CDC45 loading and origin firing [PMID:20116089, PMID:21700459, PMID:21646402]. TRESLIN functions as a stable elongated heterotetramer with MTBP, which contributes a DNA-binding C-terminal domain required for chromatin localization and CDC45 loading; reconstitution requires both proteins together, and the phospho-TRESLIN–MTBP complex is limiting for S-phase duration and origin number [PMID:28877985, PMID:25737283, PMID:34699733]. Loading of TRESLIN–MTBP onto phosphorylated MCM2-7 double hexamers is driven by DDK and opposed by RIF1–PP1, an antagonism that defines initiation zones and replication timing, with CDK cooperating with DDK to drive the TopBP1 interaction at the pre-IC step [PMID:41331242, PMID:34699733]. TRESLIN abundance and activity are tightly controlled across the cell cycle by CRL4-DTL- and PCNA-dependent proteasomal degradation upon S-phase entry, which moderates active origin number [PMID:34534348, PMID:bio_10.1101_2025.06.10.657920]. The interaction is negatively regulated during replication stress by Chk1, which binds a C-terminal TRESLIN domain (TRCT), directly phosphorylates TRESLIN, and reduces its CDK-site phosphorylation to dissociate TopBP1 and suppress origin firing; loss of Chk1–TRESLIN binding elevates origin firing even in an unperturbed S phase [PMID:25557548, PMID:21700459, PMID:35231445]. TRESLIN additionally couples replication initiation to checkpoint signaling by stimulating TopBP1-dependent ATR-mediated Chk1 phosphorylation, and through dynamic, transient action at origins it implements an ATR/Chk1-independent monitoring system that prevents premature G2 entry [PMID:23696651, PMID:36049481].","teleology":[{"year":2010,"claim":"Established TRESLIN as an essential replication factor acting at the pre-IC step, answering what links origin licensing to helicase activation.","evidence":"Immunodepletion/add-back in Xenopus egg extracts, Co-IP, siRNA knockdown and chromatin fractionation in human/zebrafish cells","pmids":["20116089","20080954"],"confidence":"High","gaps":["Molecular basis of the TopBP1 interaction not yet defined","How CDK activity bridges TRESLIN to TopBP1 unknown"]},{"year":2011,"claim":"Identified the CDK2-cyclin E phosphorylation sites on TRESLIN as the molecular switch enabling TopBP1 BRCT binding, explaining how CDK activity licenses initiation.","evidence":"In vitro kinase assay, site-directed mutagenesis of CDK sites, Co-IP and replication rescue in Xenopus and human cells","pmids":["21700459","21646402"],"confidence":"High","gaps":["Stoichiometry and structure of the phospho-TRESLIN–TopBP1 interface not resolved","Whether additional kinases contribute not addressed"]},{"year":2013,"claim":"Showed TRESLIN is dual-function, stimulating ATR-mediated Chk1 phosphorylation in a TopBP1-dependent manner, linking initiation to checkpoint signaling.","evidence":"In vitro ATR kinase assay, cell-based checkpoint assays, phospho-site mutagenesis","pmids":["23696651"],"confidence":"Medium","gaps":["Single lab, not independently confirmed","Mechanism by which Ser-1000 status modulates ATR signaling unclear"]},{"year":2014,"claim":"Defined how Chk1 docks onto TRESLIN to restrain origin firing in unperturbed S phase, revealing a built-in negative regulator of CDC45 loading.","evidence":"Co-IP, TRCT domain mutagenesis abolishing binding, EdU and CDC45 chromatin-loading assays","pmids":["25557548"],"confidence":"High","gaps":["Which TRESLIN residues Chk1 phosphorylates not yet mapped here","Relationship to CDK-site phosphorylation not resolved"]},{"year":2017,"claim":"Established MTBP as the obligate TRESLIN partner and DNA-binding module required for chromatin loading and CDC45 recruitment.","evidence":"Immunodepletion/add-back reconstitution in Xenopus extracts, DNA-binding assays, CTM domain mutagenesis, human S-phase assays","pmids":["28877985"],"confidence":"High","gaps":["Structural basis of TRESLIN–MTBP assembly not defined","DNA sequence preference of CTM in vivo not established"]},{"year":2017,"claim":"Demonstrated multiple layers controlling TRESLIN abundance and CDK-driven activity (Gwl/Ensa/PP2A-B55 degradation, CKS-enhanced phosphorylation, and mutant-p53 bypass), framing TRESLIN as a tightly tuned limiting factor.","evidence":"siRNA knockdown with rescue, proteasome inhibition, in vitro kinase assays with CKS, Co-IP and BrdU assays with p53 mutants and CDK2 inhibitor","pmids":["28785014","28739856","28439015"],"confidence":"Medium","gaps":["Each mechanism from a single lab","How these regulatory inputs are integrated in vivo unclear"]},{"year":2015,"claim":"Showed that phosphomimetic TRESLIN shortens S phase by increasing initiation in an MTBP-interaction-dependent manner, establishing phospho-TRESLIN as rate-limiting for S-phase progression.","evidence":"Phosphomimetic TICRR-TESE overexpression, EdU incorporation, DNA fiber and replication cluster analysis in human cells","pmids":["25737283"],"confidence":"Medium","gaps":["Single lab","Whether endogenous phospho-occupancy is limiting not directly measured"]},{"year":2020,"claim":"Mapped genome-wide TRESLIN-MTBP occupancy, linking the complex to open chromatin and regulatory elements rather than random origins.","evidence":"MTBP ChIP-seq with chromatin accessibility and histone modification correlation in human cells","pmids":["32966791"],"confidence":"Medium","gaps":["Whether all binding sites correspond to firing origins unknown","Functional consequence of regulatory-element binding not established"]},{"year":2021,"claim":"Resolved complex stoichiometry (2:2 tetramer) and established that DDK strengthens TRESLIN-MTBP chromatin loading and cooperates with CDK for TopBP1 engagement.","evidence":"Immunodepletion/add-back in Xenopus extracts, biochemical stoichiometry, kinase inhibitor treatments","pmids":["34699733"],"confidence":"Medium","gaps":["High-resolution structure of the tetramer not determined","DDK substrate site driving loading not mapped here"]},{"year":2021,"claim":"Identified CRL4-DTL-dependent S-phase degradation of TICRR as a mechanism limiting active origin number.","evidence":"Cell-cycle synchronization, immunoblotting, siRNA screen of E3 components, proteasome inhibition","pmids":["34534348"],"confidence":"Medium","gaps":["Degron and ubiquitination sites not mapped","Single lab"]},{"year":2022,"claim":"Detailed how Chk1 dissociates TopBP1 from TRESLIN during stress via combined CDK-activity reduction (T968) and direct phosphorylation (S1114), refining the negative-regulatory mechanism.","evidence":"Phospho-specific antibodies, CDK/Chk1 inhibitors, mutagenesis, origin firing assays in cancer cells","pmids":["35231445"],"confidence":"Medium","gaps":["Relative contribution of each phospho-event in vivo not quantified","Single lab"]},{"year":2022,"claim":"Revealed TRESLIN-MTBP acts transiently at origins and operates an ATR/CHK1-independent surveillance of origin-firing dynamics that governs the S/G2 transition.","evidence":"Live-cell imaging, chromatin fractionation, ATR/CHK1 inhibitors, knockdowns in human cells","pmids":["36049481"],"confidence":"Medium","gaps":["Molecular sensor mechanism not defined","Single lab"]},{"year":2022,"claim":"Provided a domain architecture for TRESLIN, assigning a Sld3-homologous core (Ku70-like M domain, Sld3-Treslin domain, TopBP1-interaction domain) required for origin firing.","evidence":"Sequence analysis, structural homology modeling, domain mutant expression with replication assays in human cells","pmids":["35091422"],"confidence":"Low","gaps":["Structural assignment is computational, not experimentally solved","Single lab"]},{"year":2025,"claim":"Established that DDK vs RIF1-PP1 phosphorylation of MCM-DH controls TRESLIN-MTBP loading to set initiation zones and replication timing, and that G1 chromatin binding is licensing-independent with distinct G1 vs S binding modes.","evidence":"Genome-wide TRESLIN-MTBP and MCM mapping, kinase/phosphatase inhibitors, Cut&Run, Geminin-based MCM depletion, siRNA in human cells","pmids":["41331242","40624716"],"confidence":"Medium","gaps":["Function of licensing-independent G1 binding unknown","Structural basis of phospho-MCM recognition not defined"]},{"year":2025,"claim":"Linked CDK-sensitive, PCNA-dependent TRESLIN degradation to dormant origin firing under WEE1 inhibition, identifying a conserved degron-like sequence governing this control.","evidence":"WEE1/CDK inhibitor epistasis, PCNA dependency assays, chromatin fractionation, DNA fiber analysis, domain mutagenesis (preprint)","pmids":["bio_10.1101_2025.06.10.657920"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Precise degron residues and ubiquitin ligase coupling to PCNA unresolved"]},{"year":null,"claim":"How TRESLIN-MTBP physically recognizes the phosphorylated MCM2-7 double hexamer and assembles CDC45 at atomic resolution, and how its multiple regulatory inputs are integrated to select specific origins, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental high-resolution structure of TRESLIN-MTBP or its origin complexes","Mechanism of origin-site selection from genome-wide binding not fully defined","Integration of degradation, checkpoint, and kinase inputs into a single quantitative model lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2,7]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[7,12]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,1,7,12]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,11]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0,1,2,7,17]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,11,13]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[4,5,14]}],"complexes":["TRESLIN-MTBP complex"],"partners":["MTBP","TOPBP1","CHEK1","CDK2","DTL","CKS1B","DUE-B/UBE2V2","TP53"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7Z2Z1","full_name":"Treslin","aliases":["TopBP1-interacting checkpoint and replication regulator","TopBP1-interacting, replication-stimulating protein"],"length_aa":1910,"mass_kda":210.9,"function":"Regulator of DNA replication and S/M and G2/M checkpoints. Regulates the triggering of DNA replication initiation via its interaction with TOPBP1 by participating in CDK2-mediated loading of CDC45L onto replication origins. Required for the transition from pre-replication complex (pre-RC) to pre-initiation complex (pre-IC). Required to prevent mitotic entry after treatment with ionizing radiation","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q7Z2Z1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TICRR","classification":"Common Essential","n_dependent_lines":1207,"n_total_lines":1208,"dependency_fraction":0.9991721854304636},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"MIF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TICRR","total_profiled":1310},"omim":[{"mim_id":"613298","title":"TOPBP1-INTERACTING CHECKPOINT AND REPLICATION REGULATOR; TICRR","url":"https://www.omim.org/entry/613298"},{"mim_id":"173870","title":"POLY(ADP-RIBOSE) POLYMERASE 1; PARP1","url":"https://www.omim.org/entry/173870"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"bone marrow","ntpm":3.2},{"tissue":"esophagus","ntpm":4.0},{"tissue":"lymphoid tissue","ntpm":3.9}],"url":"https://www.proteinatlas.org/search/TICRR"},"hgnc":{"alias_symbol":["MGC45866","FLJ41618","Treslin","SLD3"],"prev_symbol":["C15orf42"]},"alphafold":{"accession":"Q7Z2Z1","domains":[{"cath_id":"3.40.50.410","chopping":"2-117_141-256_285-291","consensus_level":"medium","plddt":73.4459,"start":2,"end":291},{"cath_id":"2.40.290.10","chopping":"300-437","consensus_level":"medium","plddt":69.3522,"start":300,"end":437},{"cath_id":"-","chopping":"639-809","consensus_level":"high","plddt":82.4331,"start":639,"end":809}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z2Z1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z2Z1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7Z2Z1-F1-predicted_aligned_error_v6.png","plddt_mean":47.66},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TICRR","jax_strain_url":"https://www.jax.org/strain/search?query=TICRR"},"sequence":{"accession":"Q7Z2Z1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7Z2Z1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7Z2Z1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7Z2Z1"}},"corpus_meta":[{"pmid":"17167417","id":"PMC_17167417","title":"Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast.","date":"2006","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/17167417","citation_count":390,"is_preprint":false},{"pmid":"17167415","id":"PMC_17167415","title":"CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast.","date":"2006","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/17167415","citation_count":374,"is_preprint":false},{"pmid":"20835227","id":"PMC_20835227","title":"Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation.","date":"2010","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/20835227","citation_count":227,"is_preprint":false},{"pmid":"20116089","id":"PMC_20116089","title":"Treslin collaborates with TopBP1 in triggering the initiation of DNA 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Depletion of Treslin strongly inhibits chromosomal DNA replication. Treslin binds chromatin independently of TopBP1, but Treslin-TopBP1 association requires Cdk2 activity and is necessary for Cdc45 loading.\",\n      \"method\": \"Immunodepletion from Xenopus egg extracts, co-immunoprecipitation, siRNA knockdown in human cells, chromatin fractionation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, depletion/add-back in egg extracts, corroborated by human cell knockdown, multiple orthogonal methods in one study\",\n      \"pmids\": [\"20116089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TICRR (ticrr) associates with TopBP1 via BRCT motifs essential for TopBP1's replication and checkpoint functions, and ticrr deficiency disrupts chromatin binding of pre-initiation complex (pre-IC) components without affecting pre-replication complex (pre-RC) loading, placing TICRR at the pre-IC assembly step.\",\n      \"method\": \"Zebrafish genetic screen, co-immunoprecipitation, chromatin fractionation, morpholino knockdown\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic screen plus biochemical epistasis (pre-RC vs pre-IC components), replicated in zebrafish and human cells\",\n      \"pmids\": [\"20080954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Treslin/TICRR contains two conserved CDK phosphorylation sites (orthologous to yeast Sld3 CDK sites) that are essential for DNA replication. Phosphorylation of these sites mediates interaction with the N-terminal pair of BRCT repeats in TopBP1 (ortholog of yeast Dpb11). Replication stress prevents this interaction via the Chk1 checkpoint kinase.\",\n      \"method\": \"Mutagenesis of CDK sites, co-immunoprecipitation, siRNA knockdown with replication assays, sequence conservation analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis of key CDK sites combined with Co-IP, replication assays, and checkpoint epistasis; independently supported by multiple labs\",\n      \"pmids\": [\"21700459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Treslin is phosphorylated by Cdk2-cyclin E at a conserved CDK consensus site (S976 in Xenopus, S1000 in humans). This phosphorylation is required for effective association with TopBP1 and for DNA replication. A non-phosphorylatable mutant shows severely diminished TopBP1 binding and replication deficiency.\",\n      \"method\": \"In vitro kinase assay with recombinant Cdk2-cyclin E, site-directed mutagenesis, co-immunoprecipitation, human cell replication assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus functional rescue, replicated in both Xenopus and human cells\",\n      \"pmids\": [\"21646402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Chk1 associates specifically with a C-terminal domain of Treslin (TRCT domain). This interaction enables Chk1-catalyzed phosphorylation of Treslin. Abolishing the Treslin-Chk1 interaction results in elevated replication initiation during an unperturbed S phase due to enhanced Cdc45 loading, revealing a Chk1 role in suppressing origin firing during normal S phase.\",\n      \"method\": \"Co-immunoprecipitation, TRCT domain mutagenesis, EdU incorporation assays, Cdc45 chromatin loading analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mutagenesis abolishing binding combined with functional readout (Cdc45 loading, origin firing), multiple orthogonal methods\",\n      \"pmids\": [\"25557548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Treslin stimulates ATR-mediated phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner, and the phosphorylation state of Treslin at Ser-1000 is important for this checkpoint activity, indicating Treslin is a dual replication/checkpoint protein.\",\n      \"method\": \"In vitro ATR kinase assay, cell-based checkpoint assays, phosphorylation site mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Weak — in vitro kinase assay and mutagenesis from single lab, single study\",\n      \"pmids\": [\"23696651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Overexpression of a phosphomimetic TICRR mutant (TICRR-TESE, mimicking CDK phosphorylation at two key residues) shortens S phase by increasing replication initiation. This effect requires the TICRR region necessary for interaction with MTBP (Mdm2-binding protein), demonstrating that phosphorylated TICRR is limiting for S-phase progression.\",\n      \"method\": \"Phosphomimetic mutant overexpression, EdU incorporation, DNA fiber analysis, replication cluster analysis in human cells\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphomimetic mutant with multiple replication readouts and domain requirement, single lab with orthogonal methods\",\n      \"pmids\": [\"25737283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MTBP (Mdm2-binding protein) is a binding partner of Treslin. Depletion of MTBP from Xenopus egg extracts co-depletes Treslin and abolishes DNA replication, which can only be rescued by recombinant Treslin-MTBP together, not either protein alone. MTBP contains a C-terminal domain (CTM) that binds double-stranded and G-quadruplex DNA, and CTM mutants are defective for chromatin localization and Cdc45 loading.\",\n      \"method\": \"Immunodepletion and add-back in Xenopus egg extracts, DNA-binding assays, chromatin fractionation, human cell S-phase assays, domain mutagenesis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reconstitution-like add-back experiment, mutagenesis, DNA-binding assay, functional validation in Xenopus and human cells\",\n      \"pmids\": [\"28877985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ensa (a substrate of Greatwall kinase) controls Treslin protein levels via the ubiquitin-proteasome pathway. Ensa knockdown reduces Treslin levels and extends S phase; this phenotype is rescued by Treslin overexpression. The Gwl/Ensa/PP2A-B55 pathway thus regulates S-phase duration by controlling Treslin degradation.\",\n      \"method\": \"siRNA knockdown, proteasome inhibitor treatment, rescue experiments with Treslin overexpression, flow cytometry\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — knockdown plus rescue with functional readout, single lab, two orthogonal methods\",\n      \"pmids\": [\"28785014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Mutant p53 (contact-type, e.g. R273H) enhances the interaction between TopBP1 and Treslin and promotes DNA replication even when Cdk2 is inhibited, bypassing the normal CDK requirement for Treslin-TopBP1 interaction.\",\n      \"method\": \"Co-immunoprecipitation, BrdU incorporation assays, CDK2 inhibitor treatment, expression of specific p53 mutants\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP plus functional replication assay, single lab with two orthogonal methods\",\n      \"pmids\": [\"28439015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Treslin-MTBP is rate-limiting for replication initiation. DDK activity both increases and strengthens the interaction of Treslin-MTBP with licensed chromatin, and cooperates with CDK to drive Treslin-MTBP interaction with TopBP1 at the pre-IC formation step.\",\n      \"method\": \"Immunodepletion and add-back in Xenopus egg extracts, biochemical characterization of complex stoichiometry, kinase inhibitor treatments\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — depletion/add-back plus kinase inhibitor experiments in Xenopus system, single lab\",\n      \"pmids\": [\"34699733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TICRR/TRESLIN protein levels are high in G1 and decrease upon S-phase entry via CRL4-DTL E3 ubiquitin ligase-dependent proteasomal degradation. This mechanism moderates TICRR levels to control the number of active origins during S phase.\",\n      \"method\": \"Cell cycle synchronization, immunoblotting, siRNA screens targeting E3 ligase components, proteasome inhibitor treatment\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — targeted siRNA screen identifying CRL4-DTL, proteasome inhibitor validation, cell cycle fractionation; single lab\",\n      \"pmids\": [\"34534348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MTBP (and hence the Treslin-MTBP complex) binds to at least 30,000 sites in the human genome, preferentially at regions of open chromatin containing transcriptional-regulatory elements (promoters, enhancers, super-enhancers) with nucleosome-free DNA (G-quadruplex or AP-1 motifs) and H3K4me2-marked nucleosomes.\",\n      \"method\": \"ChIP-seq genome-wide mapping of MTBP in human cells, correlation with chromatin accessibility and histone modification datasets\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — genome-wide ChIP-seq localization with functional context, single lab\",\n      \"pmids\": [\"32966791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRESLIN-MTBP acts transiently at pre-replication complexes to initiate origin firing and is released after CDC45 recruitment. The dynamic behavior of TRESLIN-MTBP implements a monitoring system that senses the rate of origin firing and the decline of active origins in late S phase, preventing premature entry into G2 independently of ATR/CHK1 kinases.\",\n      \"method\": \"Live-cell imaging, chromatin fractionation, cell cycle analysis, ATR/CHK1 inhibitor treatments, knockdown experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays with kinase inhibitors and knockdowns, single lab with orthogonal methods\",\n      \"pmids\": [\"36049481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Chk1 regulates the Treslin-TopBP1 interaction through multiple mechanisms during replication stress: (1) Chk1-dependent decrease in CDK activity reduces phosphorylation of Treslin T968 (but not S1000), and (2) Chk1 directly phosphorylates additional sites on Treslin including S1114, both contributing to dissociation of TopBP1 from Treslin and suppression of origin firing.\",\n      \"method\": \"Phospho-specific antibodies, CDK inhibitor and Chk1 inhibitor treatments, mutagenesis, replication origin firing assays in cancer cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-site specific analysis plus mutagenesis plus functional origin firing assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35231445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DUE-B interacts with Treslin in HeLa cells in a cell-cycle-regulated manner (peaking as cells exit G1). The conserved C-terminal domain of DUE-B is required for its binding to TopBP1, Treslin, Cdc45, and MCM2-7, and for efficient chromatin loading of Treslin, Cdc45, and TopBP1. The DUE-B-Treslin interaction is required for Cdc45 loading onto chromatin.\",\n      \"method\": \"Co-immunoprecipitation, domain mutagenesis, chromatin fractionation, cell cycle synchronization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus domain mutagenesis plus functional chromatin loading assay, single lab\",\n      \"pmids\": [\"30037903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CKS proteins (Cks1, Cks2) enhance the ability of Cdk2 to phosphorylate Treslin in vitro, promoting replication checkpoint recovery. CKS binding-defective mutants retain the ability to stimulate Treslin phosphorylation. Silencing of Cks1/Cks2 decreases Treslin phosphorylation in vivo, and CKS overexpression prevents checkpoint-dependent dephosphorylation of Treslin.\",\n      \"method\": \"In vitro kinase assay, siRNA knockdown, CKS mutant overexpression, phospho-Treslin immunoblotting\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay plus in vivo phosphorylation analysis, single lab with orthogonal in vitro and in vivo methods\",\n      \"pmids\": [\"28739856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRESLIN-MTBP is a key limiting firing factor for replication initiation in human cells. Its loading onto phosphorylated MCM2-7 double hexamer (MCM-DH) is controlled by opposing phosphorylation events on MCM-DH by DDK (promoting TRESLIN-MTBP loading) and RIF1-Protein Phosphatase 1 (opposing loading), which determines initiation zones and establishes replication timing.\",\n      \"method\": \"Genome-wide mapping of TRESLIN-MTBP and MCM, kinase/phosphatase inhibitor treatments, chromatin fractionation in human cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide functional mapping plus kinase/phosphatase epistasis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41331242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"During G1, MTBP depends on TRESLIN for proper chromatin association, but not during S phase, suggesting two distinct modes of TRESLIN-MTBP chromatin binding. Neither TRESLIN nor MTBP binding to chromatin during G1 requires licensed MCM origins.\",\n      \"method\": \"Cut&Run genomic binding mapping, cell cycle synchronization, Geminin-based MCM depletion, siRNA knockdown of TRESLIN/MTBP\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Cut&Run with cell cycle synchronization and MCM depletion controls, single lab with multiple conditions\",\n      \"pmids\": [\"40624716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Sequence analysis and structural homology modeling revealed that Treslin/TICRR contains a conserved Ku70-homologous β-barrel fold in its middle domain (M domain), and a von Willebrand factor type A domain in the CIT region. The M domain, Sld3-Treslin domain, and TopBP1/Dpb11 interaction domain together constitute the Sld3-homologous core. Domain mutants expressed in human cells indicate all three Sld3-core domains plus non-conserved terminal domains are required for proper origin firing.\",\n      \"method\": \"Protein sequence analysis, structural homology modeling, domain mutant expression in human cells with replication assays\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3-4 / Moderate — domain mutagenesis in human cells provides functional validation, but structural assignment is computational; single lab\",\n      \"pmids\": [\"35091422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"WEE1 kinase inhibition during S phase increases CDK activity, which blocks PCNA-dependent proteasomal degradation of TRESLIN and enhances chromatin association of TRESLIN and MTBP, leading to elevated helicase recruitment and dormant origin firing. A conserved sequence within TRESLIN is required for this CDK-sensitive degradation.\",\n      \"method\": \"WEE1 inhibitor treatment, CDK inhibitor reversal, PCNA dependency assays, chromatin fractionation, DNA fiber analysis in human cells, domain mutagenesis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological epistasis plus domain mutagenesis plus functional replication readouts; preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.06.10.657920\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TICRR/TRESLIN is an essential metazoan replication initiation factor (ortholog of yeast Sld3) that forms a stable complex with MTBP (ortholog of Sld7), is phosphorylated by CDK2 (at T968/S1000 in humans), and in its phosphorylated form interacts with TopBP1 BRCT repeats to mediate loading of CDC45 onto the MCM2-7 helicase and assemble the active CMG helicase at replication origins; this central interaction is negatively regulated by Chk1 (which directly phosphorylates Treslin and promotes dephosphorylation of its CDK sites during replication stress) and is subject to CRL4-DTL-mediated proteasomal degradation during S phase, while DDK activity promotes TRESLIN-MTBP loading onto phosphorylated MCM double hexamers and cooperates with CDK to drive pre-initiation complex formation, making TRESLIN-MTBP a key rate-limiting determinant of origin selection, replication timing, and the S/G2 transition.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TICRR/TRESLIN is an essential metazoan DNA replication initiation factor that acts at the pre-initiation complex (pre-IC) assembly step to load CDC45 onto licensed origins, and its depletion abolishes chromosomal DNA replication [#0, #1]. TRESLIN binds chromatin independently of TopBP1, but its association with the N-terminal BRCT repeats of TopBP1 requires CDK2-cyclin E phosphorylation at conserved CDK consensus sites (S1000/T968 in humans), and this phospho-dependent TRESLIN–TopBP1 interaction is the rate-limiting event for CDC45 loading and origin firing [#0, #2, #3]. TRESLIN functions as a stable elongated heterotetramer with MTBP, which contributes a DNA-binding C-terminal domain required for chromatin localization and CDC45 loading; reconstitution requires both proteins together, and the phospho-TRESLIN–MTBP complex is limiting for S-phase duration and origin number [#7, #6, #10]. Loading of TRESLIN–MTBP onto phosphorylated MCM2-7 double hexamers is driven by DDK and opposed by RIF1–PP1, an antagonism that defines initiation zones and replication timing, with CDK cooperating with DDK to drive the TopBP1 interaction at the pre-IC step [#17, #10]. TRESLIN abundance and activity are tightly controlled across the cell cycle by CRL4-DTL- and PCNA-dependent proteasomal degradation upon S-phase entry, which moderates active origin number [#11, #20]. The interaction is negatively regulated during replication stress by Chk1, which binds a C-terminal TRESLIN domain (TRCT), directly phosphorylates TRESLIN, and reduces its CDK-site phosphorylation to dissociate TopBP1 and suppress origin firing; loss of Chk1–TRESLIN binding elevates origin firing even in an unperturbed S phase [#4, #2, #14]. TRESLIN additionally couples replication initiation to checkpoint signaling by stimulating TopBP1-dependent ATR-mediated Chk1 phosphorylation, and through dynamic, transient action at origins it implements an ATR/Chk1-independent monitoring system that prevents premature G2 entry [#5, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established TRESLIN as an essential replication factor acting at the pre-IC step, answering what links origin licensing to helicase activation.\",\n      \"evidence\": \"Immunodepletion/add-back in Xenopus egg extracts, Co-IP, siRNA knockdown and chromatin fractionation in human/zebrafish cells\",\n      \"pmids\": [\"20116089\", \"20080954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the TopBP1 interaction not yet defined\", \"How CDK activity bridges TRESLIN to TopBP1 unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified the CDK2-cyclin E phosphorylation sites on TRESLIN as the molecular switch enabling TopBP1 BRCT binding, explaining how CDK activity licenses initiation.\",\n      \"evidence\": \"In vitro kinase assay, site-directed mutagenesis of CDK sites, Co-IP and replication rescue in Xenopus and human cells\",\n      \"pmids\": [\"21700459\", \"21646402\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structure of the phospho-TRESLIN–TopBP1 interface not resolved\", \"Whether additional kinases contribute not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed TRESLIN is dual-function, stimulating ATR-mediated Chk1 phosphorylation in a TopBP1-dependent manner, linking initiation to checkpoint signaling.\",\n      \"evidence\": \"In vitro ATR kinase assay, cell-based checkpoint assays, phospho-site mutagenesis\",\n      \"pmids\": [\"23696651\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, not independently confirmed\", \"Mechanism by which Ser-1000 status modulates ATR signaling unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined how Chk1 docks onto TRESLIN to restrain origin firing in unperturbed S phase, revealing a built-in negative regulator of CDC45 loading.\",\n      \"evidence\": \"Co-IP, TRCT domain mutagenesis abolishing binding, EdU and CDC45 chromatin-loading assays\",\n      \"pmids\": [\"25557548\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which TRESLIN residues Chk1 phosphorylates not yet mapped here\", \"Relationship to CDK-site phosphorylation not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established MTBP as the obligate TRESLIN partner and DNA-binding module required for chromatin loading and CDC45 recruitment.\",\n      \"evidence\": \"Immunodepletion/add-back reconstitution in Xenopus extracts, DNA-binding assays, CTM domain mutagenesis, human S-phase assays\",\n      \"pmids\": [\"28877985\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TRESLIN–MTBP assembly not defined\", \"DNA sequence preference of CTM in vivo not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated multiple layers controlling TRESLIN abundance and CDK-driven activity (Gwl/Ensa/PP2A-B55 degradation, CKS-enhanced phosphorylation, and mutant-p53 bypass), framing TRESLIN as a tightly tuned limiting factor.\",\n      \"evidence\": \"siRNA knockdown with rescue, proteasome inhibition, in vitro kinase assays with CKS, Co-IP and BrdU assays with p53 mutants and CDK2 inhibitor\",\n      \"pmids\": [\"28785014\", \"28739856\", \"28439015\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Each mechanism from a single lab\", \"How these regulatory inputs are integrated in vivo unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed that phosphomimetic TRESLIN shortens S phase by increasing initiation in an MTBP-interaction-dependent manner, establishing phospho-TRESLIN as rate-limiting for S-phase progression.\",\n      \"evidence\": \"Phosphomimetic TICRR-TESE overexpression, EdU incorporation, DNA fiber and replication cluster analysis in human cells\",\n      \"pmids\": [\"25737283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Whether endogenous phospho-occupancy is limiting not directly measured\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapped genome-wide TRESLIN-MTBP occupancy, linking the complex to open chromatin and regulatory elements rather than random origins.\",\n      \"evidence\": \"MTBP ChIP-seq with chromatin accessibility and histone modification correlation in human cells\",\n      \"pmids\": [\"32966791\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether all binding sites correspond to firing origins unknown\", \"Functional consequence of regulatory-element binding not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved complex stoichiometry (2:2 tetramer) and established that DDK strengthens TRESLIN-MTBP chromatin loading and cooperates with CDK for TopBP1 engagement.\",\n      \"evidence\": \"Immunodepletion/add-back in Xenopus extracts, biochemical stoichiometry, kinase inhibitor treatments\",\n      \"pmids\": [\"34699733\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"High-resolution structure of the tetramer not determined\", \"DDK substrate site driving loading not mapped here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified CRL4-DTL-dependent S-phase degradation of TICRR as a mechanism limiting active origin number.\",\n      \"evidence\": \"Cell-cycle synchronization, immunoblotting, siRNA screen of E3 components, proteasome inhibition\",\n      \"pmids\": [\"34534348\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degron and ubiquitination sites not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Detailed how Chk1 dissociates TopBP1 from TRESLIN during stress via combined CDK-activity reduction (T968) and direct phosphorylation (S1114), refining the negative-regulatory mechanism.\",\n      \"evidence\": \"Phospho-specific antibodies, CDK/Chk1 inhibitors, mutagenesis, origin firing assays in cancer cells\",\n      \"pmids\": [\"35231445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of each phospho-event in vivo not quantified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed TRESLIN-MTBP acts transiently at origins and operates an ATR/CHK1-independent surveillance of origin-firing dynamics that governs the S/G2 transition.\",\n      \"evidence\": \"Live-cell imaging, chromatin fractionation, ATR/CHK1 inhibitors, knockdowns in human cells\",\n      \"pmids\": [\"36049481\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular sensor mechanism not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided a domain architecture for TRESLIN, assigning a Sld3-homologous core (Ku70-like M domain, Sld3-Treslin domain, TopBP1-interaction domain) required for origin firing.\",\n      \"evidence\": \"Sequence analysis, structural homology modeling, domain mutant expression with replication assays in human cells\",\n      \"pmids\": [\"35091422\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Structural assignment is computational, not experimentally solved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established that DDK vs RIF1-PP1 phosphorylation of MCM-DH controls TRESLIN-MTBP loading to set initiation zones and replication timing, and that G1 chromatin binding is licensing-independent with distinct G1 vs S binding modes.\",\n      \"evidence\": \"Genome-wide TRESLIN-MTBP and MCM mapping, kinase/phosphatase inhibitors, Cut&Run, Geminin-based MCM depletion, siRNA in human cells\",\n      \"pmids\": [\"41331242\", \"40624716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Function of licensing-independent G1 binding unknown\", \"Structural basis of phospho-MCM recognition not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked CDK-sensitive, PCNA-dependent TRESLIN degradation to dormant origin firing under WEE1 inhibition, identifying a conserved degron-like sequence governing this control.\",\n      \"evidence\": \"WEE1/CDK inhibitor epistasis, PCNA dependency assays, chromatin fractionation, DNA fiber analysis, domain mutagenesis (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.10.657920\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Precise degron residues and ubiquitin ligase coupling to PCNA unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TRESLIN-MTBP physically recognizes the phosphorylated MCM2-7 double hexamer and assembles CDC45 at atomic resolution, and how its multiple regulatory inputs are integrated to select specific origins, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental high-resolution structure of TRESLIN-MTBP or its origin complexes\", \"Mechanism of origin-site selection from genome-wide binding not fully defined\", \"Integration of degradation, checkpoint, and kinase inputs into a single quantitative model lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2, 7]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [7, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 1, 7, 12]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [0, 1, 2, 7, 17]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 11, 13]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [4, 5, 14]}\n    ],\n    \"complexes\": [\"TRESLIN-MTBP complex\"],\n    \"partners\": [\"MTBP\", \"TOPBP1\", \"CHEK1\", \"CDK2\", \"DTL\", \"CKS1B\", \"DUE-B/UBE2V2\", \"TP53\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}