{"gene":"USP1","run_date":"2026-04-28T21:43:01","timeline":{"discoveries":[{"year":2005,"finding":"USP1 physically associates with FANCD2 and deubiquitinates monoubiquitinated FANCD2, with both proteins colocalizing on chromatin after DNA damage; RNAi inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2 and chromosomal aberrations, placing USP1 as a deubiquitinase in the Fanconi anemia pathway.","method":"RNAi library screen, Co-IP, colocalization, chromosomal aberration assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus functional KD phenotype, replicated widely across subsequent studies","pmids":["15694335"],"is_preprint":false},{"year":1998,"finding":"Recombinant USP1 protein possesses genuine ubiquitin-specific protease (UBP) activity, correctly cleaving a Ub-beta-galactosidase fusion to produce free ubiquitin and beta-galactosidase, identifying USP1 as an active deubiquitinating enzyme.","method":"In vitro ubiquitin cleavage assay with recombinant protein","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical reconstitution of enzymatic activity","pmids":["9806842"],"is_preprint":false},{"year":2009,"finding":"Targeted deletion of mouse Usp1 results in elevated monoubiquitinated Fancd2 in chromatin, impaired Fancd2 foci assembly, defective homologous recombination repair, and an FA-like phenotype (crosslinker hypersensitivity, male infertility, perinatal lethality); double KO of Usp1 and Fancd2 yields a more severe phenotype, placing Usp1 downstream of Fancd2 monoubiquitination in the FA pathway.","method":"Targeted gene knockout in mice, epistasis (double KO), chromatin fractionation, HR assay","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with double-KO, multiple orthogonal cellular phenotype assays","pmids":["19217432"],"is_preprint":false},{"year":2011,"finding":"USP1 binds, deubiquitinates, and thereby stabilizes the ID1, ID2, and ID3 inhibitor-of-differentiation proteins; USP1 knockdown in osteosarcoma cells leads to ID protein destabilization, cell-cycle arrest, and osteogenic differentiation, while ectopic USP1 in mesenchymal stem cells stabilizes IDs and inhibits osteoblastic differentiation.","method":"Co-IP, ubiquitination assay, siRNA knockdown, ectopic expression, in vivo mouse model","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including Co-IP, in vitro deubiquitination, and in vivo validation","pmids":["21925315"],"is_preprint":false},{"year":2010,"finding":"Human ELG1 interacts with the USP1-UAF1 complex and specifically directs USP1-UAF1 to deubiquitinate PCNA; ELG1 knockdown increases PCNA monoubiquitination without affecting FANCD2 ubiquitination, and a conserved N-terminal domain of ELG1 mediates USP1-UAF1 interaction.","method":"Co-IP, siRNA knockdown, domain mapping, ubiquitination assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple methods including Co-IP, domain mapping, and selective substrate phenotype","pmids":["20147293"],"is_preprint":false},{"year":2011,"finding":"USP1 and its binding partner UAF1 form a complex that promotes homologous recombination (HR)-mediated double-strand break repair; loss of UAF1 in chicken DT40 cells causes sensitivity to camptothecin and PARP inhibitors that is rescued by disrupting NHEJ, indicating USP1/UAF1 promotes HR by suppressing NHEJ.","method":"Genetic KO in DT40 cells, epistasis with NHEJ-deficient cells, drug sensitivity assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with multiple drug sensitivity assays and pathway rescue","pmids":["21482670"],"is_preprint":false},{"year":2011,"finding":"USP1 is degraded in G1 via APC/C(Cdh1)-mediated ubiquitination; low USP1 levels in G1 are required for PCNA monoubiquitination in response to UV damage before DNA replication, as a non-degradable USP1 mutant inhibits PCNA monoubiquitination and likely impairs translesion synthesis polymerase recruitment.","method":"Cell cycle synchronization, APC/C(Cdh1) substrate assay, USP1 mutant expression, PCNA ubiquitination assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — cell-cycle-specific degradation confirmed with non-degradable mutant and functional PCNA ubiquitination readout","pmids":["21768287"],"is_preprint":false},{"year":2011,"finding":"USP1 depletion stimulates DDB1-dependent degradation of phosphorylated CHK1; USP1 maintains both total and phosphorylated CHK1 levels in response to genotoxic stress, partly in a monoubiquitinated FANCD2-dependent manner, establishing a feedback circuit where activated CHK1 upregulates FANCD2 monoubiquitination, which then promotes USP1-mediated CHK1 degradation.","method":"siRNA knockdown, western blot, Co-IP, genotoxic stress assays","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — single lab, multiple methods but complex feedback circuit partially inferred","pmids":["21389083"],"is_preprint":false},{"year":2012,"finding":"The C-terminal autocleavage fragment of USP1 (Gln-Usp1(Ct)) is a physiological substrate of the Arg/N-end rule pathway, targeted for degradation through deamidation of its N-terminal glutamine by Ntaq1; the autocleaved fragments remain associated with UAF1 and retain deubiquitylase activity until Gln-Usp1(Ct) is selectively destroyed, and stabilization of this fragment decreases PCNA monoubiquitylation and increases UV sensitivity.","method":"Biochemical reconstitution, N-end rule substrate identification, mutant analysis, cellular UV sensitivity assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — reconstitution of N-end rule degradation plus functional cellular validation with UV sensitivity","pmids":["23159736"],"is_preprint":false},{"year":2012,"finding":"USP1 activity is stimulated through formation of a tight complex with the WD40 repeat protein UAF1; kinetic analyses show UAF1 activates USP1 by lowering the pKa of the catalytic histidine (by 0.43 pH units), facilitating general base catalysis at neutral pH without altering ubiquitin binding affinity; the catalytic triad is C90, H593, and D751.","method":"Kinetic analysis (pH-dependent inactivation, solvent isotope effects, proton inventory), in vitro biochemical assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — detailed mechanistic enzymology with kinetic parameters and active-site characterization","pmids":["22439892"],"is_preprint":false},{"year":2012,"finding":"USP1 contains two nuclear localization signals (NLSs) that mediate nuclear import of the USP1/UAF1 complex; USP1 and UAF1 form a complex in the cytoplasm that translocates to the nucleus via USP1 NLSs, and a 100 amino acid motif in USP1 mediates UAF1 binding.","method":"Nuclear relocation assay, deletion mutagenesis, cellular localization","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 — localization with functional consequence mapped by mutagenesis, single lab","pmids":["22701671"],"is_preprint":false},{"year":2013,"finding":"CAPNS1 (calpain regulatory subunit) is required for USP1 stability; calpain stabilizes USP1 by activating Cdk5, which inhibits Cdh1 and consequently prevents APC/C(Cdh1)-mediated USP1 degradation in G1; loss of CAPNS1 increases PCNA monoubiquitination and polymerase-η loading.","method":"siRNA knockdown, western blot, chromatin fractionation, cell-cycle analysis, Cdk5/p25 rescue","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — genetic manipulation with rescue experiment, single lab","pmids":["23589330"],"is_preprint":false},{"year":2014,"finding":"The selective USP1-UAF1 inhibitor ML323 inhibits the USP1-UAF1 complex with excellent selectivity; pharmacological inhibition reveals that USP1-UAF1 deubiquitination is required for the DNA translesion synthesis and Fanconi anemia pathways in response to UV- and cisplatin-induced DNA damage, and ML323 potentiates cisplatin cytotoxicity.","method":"Chemical inhibitor (ML323), cell-based PCNA and FANCD2 ubiquitination assays, selectivity profiling","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 2 — highly selective chemical probe with orthogonal substrate readouts across multiple DNA damage pathways","pmids":["24531842"],"is_preprint":false},{"year":2015,"finding":"USP1 phosphorylation at S313 is not critical for PCNA deubiquitination or UAF1 binding; amino acid motif 420-520 in USP1 is necessary and sufficient for UAF1 binding (mapping to the Fingers subdomain); USP1 autocleavage occurs in cis at the G670/G671 diglycine motif and can be reduced by cancer-associated mutation L669P.","method":"Mutagenesis (S313A, S313D, deletion mutants, GG/AA, cancer-associated mutants), cell-based UAF1 binding and PCNA deubiquitination assays","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 — systematic structure-function mutagenesis, single lab","pmids":["25744535"],"is_preprint":false},{"year":2016,"finding":"The USP1-UAF1 complex interacts with RAD51AP1 (a RAD51-interacting protein) via UAF1; USP1-UAF1 stabilizes RAD51AP1 and promotes HR repair independently of FANCD2 deubiquitination; cells expressing UAF1-interaction-deficient RAD51AP1 show increased chromosomal aberrations and persistent RAD51 foci after DNA damage.","method":"Proteomics, Co-IP, domain mapping, HR assay, RAD51 foci, chromosomal aberration assay","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 — MS interactome plus functional epistasis, single lab","pmids":["27463890"],"is_preprint":false},{"year":2016,"finding":"PDGF signaling upregulates USP1 transcription through E2F transcription factors that directly bind and activate the Usp1 promoter; USP1 then stabilizes ID2 protein, which is required for proneural glioma cell survival.","method":"Mouse model, comparative transcriptomics, ChIP (E2F binding to Usp1 promoter), USP1 KO, ID2 protein stability assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus genetic ablation with in vivo tumor model, single lab","pmids":["26951930"],"is_preprint":false},{"year":2017,"finding":"The USP1-UAF1 complex interacts with TBK1, removes its K48-linked polyubiquitination, and prevents TBK1 proteasomal degradation, thereby stabilizing TBK1 and enhancing TLR3/4- and RIG-I-induced IRF3 activation and IFN-β secretion during antiviral responses.","method":"Co-IP, ubiquitination assay, KD/KO, IFN-β reporter assay, in vivo viral infection model","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional deubiquitination readout with in vivo validation, single lab","pmids":["29138248"],"is_preprint":false},{"year":2018,"finding":"USP1 binds to and is stimulated by fork DNA; a truncated USP1 lacking its DNA-binding region is not stimulated by DNA, fails to localize to replication forks, and cannot protect replication forks; persistence of monoubiquitinated PCNA at the replication fork is the mechanism of cell death when USP1 is absent in BRCA1-deficient cells.","method":"DNA-binding assay, truncation mutants, replication fork protection assay, PCNA-Ub localization, cell viability","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — multiple mechanistic assays with defined truncation mutants and specific cellular phenotype in BRCA1-deficient cells","pmids":["30576655"],"is_preprint":false},{"year":2018,"finding":"USP1 deubiquitinates and stabilizes KPNA2 (importin subunit alpha-2) protein; USP1-mediated KPNA2 stabilization is the downstream event crucial for USP1's pro-metastatic function in breast cancer cells.","method":"Co-IP, ubiquitination assay, siRNA/inhibitor-based KD, metastasis assay in mice","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP and deubiquitination assay with in vivo metastasis readout, single lab","pmids":["30531833"],"is_preprint":false},{"year":2018,"finding":"USP1 interacts with and deubiquitinates ULK1 via K63-linked ubiquitin chains; USP1 depletion or inhibition causes ULK1 sequestration to an insoluble/aggresome fraction and inhibits canonical autophagy while promoting an alternative lysosomal-mediated degradation route.","method":"Co-IP, ubiquitination linkage assay, cell fractionation, immunofluorescence, autophagic flux assay","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with K63 linkage specificity and subcellular localization with functional consequence, single lab","pmids":["30335599"],"is_preprint":false},{"year":2018,"finding":"USP1 positively regulates protein stability of ID1 and CHK1 in glioblastoma stem-like cells, and USP1 inhibition attenuates clonogenic growth and enhances radiosensitivity.","method":"shRNA knockdown, pimozide inhibitor, protein stability assay, clonogenic assay, in vivo mouse model","journal":"Neuro-oncology","confidence":"Medium","confidence_rationale":"Tier 3 — KD with protein stability readout, single lab","pmids":["26032834"],"is_preprint":false},{"year":2019,"finding":"Upon platinum treatment, USP1 is phosphorylated by ATM and ATR, binds to Snail, deubiquitinates and stabilizes Snail expression, conferring platinum resistance and promoting tumor dissemination; USP1 KO or inhibition increased platinum sensitivity in a Snail-dependent manner.","method":"Co-IP, in vivo ubiquitination assay, ATM/ATR phosphorylation assay, rescue experiments with Snail overexpression, in vivo mouse model","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — multiple biochemical methods with in vivo validation, single lab","pmids":["31086816"],"is_preprint":false},{"year":2019,"finding":"Efficient FANCD2 deubiquitination by the USP1-UAF1 complex requires DNA and is dependent on DNA binding by UAF1; RAD51AP1, via its own DNA-binding activity, can substitute for UAF1 DNA binding in FANCD2 deubiquitination in reconstituted biochemical systems and in cells.","method":"Reconstituted biochemical deubiquitination assay, DNA-binding mutants of UAF1 and RAD51AP1, cellular FANCD2 deubiquitination assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — reconstituted biochemical system with mutagenesis and cellular validation","pmids":["31253762"],"is_preprint":false},{"year":2019,"finding":"TonEBP sequentially recruits E3 ubiquitin ligase SHPRH and then USP1 to DNA damage sites, coordinating PCNA polyubiquitination dynamics; the Rel-homology domain of TonEBP is essential for interaction with both SHPRH and USP1.","method":"Co-IP, DNA damage recruitment assay, domain mapping, PCNA polyubiquitination assay","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and recruitment assay, single lab","pmids":["31376680"],"is_preprint":false},{"year":2020,"finding":"USP1 removes K63-linked polyubiquitin chains on Akt to restrict PI3K-Akt-FoxO signaling in mouse muscle during prolonged starvation; Dab2 is essential for recruiting Akt to a USP1-TSC1-PHLPP1 complex for deubiquitination, and USP1 depletion increases Akt ubiquitination and glucose uptake during fasting.","method":"DUB screening platform, Co-IP, mass spectrometry, ubiquitination (K63-linkage) assay, in vivo mouse muscle USP1 depletion","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 — DUB screen plus Co-IP/MS complex identification and in vivo validation, single lab","pmids":["32133736"],"is_preprint":false},{"year":2020,"finding":"USP1 interacts with and deubiquitinates ERα (estrogen receptor alpha), inhibiting K48-linked poly-ubiquitination and thereby stabilizing ERα protein in breast cancer cells.","method":"DUB siRNA library screen, Co-IP, ubiquitination assay","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 3 — DUB screen plus Co-IP with ubiquitination assay, single lab","pmids":["33123289"],"is_preprint":false},{"year":2020,"finding":"USP1 maintains USP1 protein stability in part by limiting TSC1 levels to sustain mTOR-mediated basal protein synthesis that maintains USP1's own levels, creating an auto-regulatory loop.","method":"Co-IP, siRNA knockdown, protein stability assay","journal":"EMBO reports","confidence":"Low","confidence_rationale":"Tier 3 — supporting biochemical data within a broader study, single lab","pmids":["32133736"],"is_preprint":false},{"year":2021,"finding":"The crystal structure of human USP1-UAF1 with and without ubiquitin, and a cryo-EM structure of USP1-UAF1 bound to monoubiquitinated FANCI-FANCD2, reveal that UAF1 drives conformational changes in FANCI-FANCD2 for deubiquitination; an extensive UAF1-FANCI interface (confirmed by mutagenesis) explains why both UAF1 and FANCI are required despite neither being directly catalytic.","method":"X-ray crystallography, cryo-EM, mutagenesis, biochemical deubiquitination assay","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure + cryo-EM + mutagenesis + biochemical validation in a single study","pmids":["33795880"],"is_preprint":false},{"year":2021,"finding":"The N-terminus of USP1 contains a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2; the N-terminus is not required for PCNA or FANCI deubiquitination, and the N-terminus alone is sufficient to confer FANCD2 specificity on a more promiscuous USP.","method":"Reconstituted deubiquitination assay with purified monoubiquitinated substrates, N-terminal deletion/chimera mutants","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 1 — biochemical reconstitution with defined substrates and domain-swap/chimera experiments","pmids":["30456385"],"is_preprint":false},{"year":2021,"finding":"Insert L1 of USP1 limits intrinsic USP1 activity and is required for both UAF1-mediated allosteric activation and substrate-dependent (DNA and PCNA) activity enhancement independently of UAF1; insert L3 also contributes to limiting intrinsic activity.","method":"Biochemical activity assays with insert deletion mutants, UAF1 activation assays, DNA/PCNA stimulation assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with multiple mutants defining allosteric mechanism","pmids":["33619839"],"is_preprint":false},{"year":2021,"finding":"GRP75 forms a complex with USP1 and SIX1, recruiting USP1 to inhibit K48-linked polyubiquitination of SIX1; GRP75's C-terminus (433-679 aa) peptide-binding domain is required for GRP75-USP1-SIX1 complex formation.","method":"Co-IP, domain mapping, ubiquitination assay, xenograft model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with domain mapping and functional readout, single lab","pmids":["34079090"],"is_preprint":false},{"year":2021,"finding":"USP1 interacts with and deubiquitinates BCAT2 at K229; BCAA increases USP1 protein levels via the GCN2-eIF2α pathway, and USP1-mediated BCAT2 stabilization promotes PDAC development.","method":"DUB library screen, Co-IP, in vitro deubiquitination assay, K229 site mapping, GCN2-eIF2α pathway analysis","journal":"National science review","confidence":"Medium","confidence_rationale":"Tier 2 — DUB screen plus in vitro deubiquitination with site identification, single lab","pmids":["35663242"],"is_preprint":false},{"year":2021,"finding":"USP1 interacts with and deubiquitinates RPS16, removing K48-linked ubiquitin chains to prevent proteasomal degradation; catalytically inactive USP1-C90A mutant cannot stabilize RPS16, confirming USP1 enzymatic activity is required.","method":"Mass spectrometry, Co-IP, western blot, C90A mutant, ubiquitination assay, xenograft model","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with active-site mutant control and in vivo validation, single lab","pmids":["34154657"],"is_preprint":false},{"year":2021,"finding":"USP1 deubiquitinates Aurora B to prevent its degradation, mediating glucocorticoid resistance in T-ALL; ALKBH5-mediated m6A demethylation increases USP1 mRNA stability, thereby elevating USP1 and Aurora B levels.","method":"Co-IP, deubiquitination assay, siRNA knockdown, rescue experiments, in vivo mouse model","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus functional rescue, single lab","pmids":["34169564"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM at 2.5-Å resolution of USP1 with and without ML323 reveals that ML323 binds to a cryptic hydrophobic site within USP1, disrupts part of the hydrophobic core, and causes conformational changes in secondary structure that lead to subtle rearrangements in the active site underlying inhibition.","method":"Cryo-EM (2.5 Å resolution), structure-based mechanistic analysis","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structure with mechanistic interpretation of inhibitor binding mode","pmids":["36170365"],"is_preprint":false},{"year":2022,"finding":"Cells harboring an autocleavage-defective USP1 mutant experience more replication fork-stalling and premature fork termination; USP1 molecules are removed from active DNA synthesis sites by the DNA-dependent metalloprotease Spartan to prevent 'USP1-trapping' lesions; autocleavage enables USP1 recycling from DNA.","method":"Autocleavage-defective mutant, super-resolution microscopy, live-cell single-molecule tracking, replication fork assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — live-cell single-molecule imaging plus replication assay with defined mutant and Spartan genetic interaction","pmids":["35365626"],"is_preprint":false},{"year":2022,"finding":"USP1 inhibition leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage; CRISPR screens identify RAD18 and UBE2K (promoting PCNA mono- and polyubiquitination) as required mediators of USP1 dependency; ectopic expression of WT but not K164R PCNA reverses USP1 inhibitor sensitivity, placing USP1 dependency on aberrant processing of ubiquitinated PCNA.","method":"CRISPR-Cas9 genome-wide screens, PCNA ubiquitination assay, ectopic PCNA expression (WT vs K164R mutant), DNA synthesis assay","journal":"Molecular cancer therapeutics","confidence":"High","confidence_rationale":"Tier 2 — genome-wide CRISPR screen validated by PCNA mutant rescue experiment","pmids":["36228090"],"is_preprint":false},{"year":2022,"finding":"USP1 interacts with and deubiquitinates MAST1, preventing its K48-linked polyubiquitination and extending its half-life; USP1-stabilized MAST1 activates MEK1, driving cisplatin resistance.","method":"CRISPR/Cas9-based genome-wide dual screen, Co-IP, domain mapping, in vitro deubiquitination assay, half-life analysis, xenograft model","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR screen plus in vitro deubiquitination assay with in vivo validation, single lab","pmids":["35966591"],"is_preprint":false},{"year":2022,"finding":"USP1 interacts with and deubiquitinates TAZ via the Hippo pathway in osteosarcoma; USP1 inhibition by ML323 destabilizes TAZ, reducing nuclear accumulation and expression of Hippo downstream genes, suppressing OS growth and metastasis in vitro and in vivo.","method":"Co-IP, ubiquitination assay, in vitro and in vivo tumor models","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus functional readout, single lab","pmids":["35637948"],"is_preprint":false},{"year":2022,"finding":"USP1 interacts with and deubiquitinates MAX, maintaining its stability; USP1-stabilized MAX promotes MYC target gene transcription, contributing to chemotherapy resistance in DLBCL.","method":"Co-IP, ubiquitination assay, shRNA/pimozide inhibition, MYC target gene expression, xenograft and PDX models","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with functional rescue and in vivo validation, single lab","pmids":["36352191"],"is_preprint":false},{"year":2023,"finding":"USP1 deubiquitinates and stabilizes TAZ (WWTR1) in CD4+ T cells, enhancing RORγt activity and Th17 differentiation while promoting Foxp3 proteasomal degradation to suppress Treg differentiation; ML323 inhibition of USP1 shifts this balance toward Treg differentiation in vitro and in vivo.","method":"siRNA screen, Co-IP, ubiquitination assay, Th17/Treg differentiation assays, in vivo inflammatory model","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — DUB screen plus Co-IP and in vivo validation, single lab","pmids":["36600049"],"is_preprint":false},{"year":2023,"finding":"The USP1-UAF1 complex interacts with and stabilizes METTL3 by removing K48-linked ubiquitination after spinal cord injury, and this stabilization promotes reactive astrogliosis through METTL3-mediated m6A methylation of YAP1 mRNA.","method":"Co-IP, ubiquitination assay, METTL3 conditional KO, m6A-seq, RNA stability assay, in vivo SCI model","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with K48-linkage specificity and in vivo rescue, single lab","pmids":["36653190"],"is_preprint":false},{"year":2023,"finding":"USP1 interacts with and deubiquitinates PARP1, removing the ubiquitin chain at K197 to prevent its proteasomal degradation; GCN5 acetyltransferase acetylates USP1 at K130, enhancing the USP1-PARP1 interaction and further increasing PARP1 stabilization.","method":"Proteomics, ubiquitylome analysis, Co-IP, GST pull-down, immunofluorescence, deletion mutant mapping, acetyltransferase assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — proteomics-guided Co-IP plus ubiquitylome with site mapping and PTM writer identification, single lab","pmids":["37821462"],"is_preprint":false},{"year":2023,"finding":"USP1 interacts with PLK1 and deubiquitinates it; USP1-stabilized PLK1 promotes LDHA expression and aerobic glycolysis in T-ALL cells.","method":"Co-IP, deubiquitination assay, siRNA knockdown, rescue experiments with LDHA","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with functional rescue, single lab","pmids":["36912760"],"is_preprint":false},{"year":2023,"finding":"USP1 interacts with and deubiquitinates C/EBPβ, increasing its protein stability; catalytically inactive USP1 has no effect on C/EBPβ stability; USP1-mediated C/EBPβ stabilization promotes adipogenesis and lipid accumulation.","method":"Co-IP, ubiquitination assay, USP1 C90A catalytic mutant, adipogenic differentiation assay, HFD mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with catalytic mutant control and in vivo HFD validation, single lab","pmids":["38012162"],"is_preprint":false},{"year":2023,"finding":"USP1 interacts with the WW domain of TAZ in HCC and stabilizes it by suppressing K11-linked polyubiquitination; a DUB siRNA screen identified USP1 as a critical regulator of Hippo/TAZ signaling activity.","method":"DUB siRNA screen, Co-IP, ubiquitination linkage assay, RNA-seq, xenograft model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — DUB screen plus Co-IP with specific K11-linkage identification, single lab","pmids":["37041150"],"is_preprint":false},{"year":2023,"finding":"Human cytomegalovirus UL138 interacts with the UAF1-USP1 complex; this interaction activates STAT1 phosphorylation (pSTAT1), and USP1 inhibition disrupts HCMV latency establishment, increasing viral replication.","method":"Co-IP, pSTAT1 assay, USP1 inhibitor (ML323), viral replication assay, ChIP of pSTAT1 on viral genome","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with functional inhibitor readout in viral infection model, single lab","pmids":["37289831"],"is_preprint":false},{"year":2023,"finding":"USP1 inhibitor ML323 stabilizes NLRP3 and AIM2 by deubiquitination in thyroid follicular cells, and USP1 modulates NLRP3 transcription by facilitating p65 nuclear transport; USP1 knockdown reduces pyroptosis markers in vitro and ameliorates HT in vivo.","method":"Co-IP, ubiquitination assay, KD/inhibitor, pyroptosis assay, p65 nuclear translocation assay, in vivo HT mouse model","journal":"Molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus multiple functional assays, single lab","pmids":["39134949"],"is_preprint":false},{"year":2024,"finding":"USP1 deubiquitinates PARP1 by removing K63-linked polyubiquitination, controlling PARP1 chromatin trapping and PARylation activity, thereby regulating sensitivity to PARP inhibitors; combined USP1/PARP1 blockade enhances replicative stress and cell death.","method":"Co-IP, K63-ubiquitination assay, PARP trapping assay, PARylation assay, cell death assay","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with K63-linkage specificity and multiple functional readouts (trapping, PARylation), single lab","pmids":["39536107"],"is_preprint":false},{"year":2024,"finding":"USP1 promotes PRIMPOL-generated ssDNA gap accumulation and their bidirectional expansion by MRE11 and EXO1 nucleases during replication stress; this activity is linked to USP1's role in PCNA deubiquitination, as PCNA ubiquitination prevents gap accumulation.","method":"siRNA depletion, ssDNA gap assay, nuclease inhibitor assays, PCNA ubiquitination assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional assays with defined mechanistic link to PCNA deubiquitination, single lab","pmids":["38180818"],"is_preprint":false},{"year":2024,"finding":"USP1 inhibition promotes ssDNA gap accumulation at replication forks via RAD18-dependent PCNA monoubiquitination; knockdown of RAD18 causes USP1 inhibitor resistance and suppresses ssDNA gap formation, establishing this as the mechanism of USP1 inhibitor-induced synthetic lethality in BRCA1-deficient cells.","method":"ssDNA gap assay, RAD18 KD rescue, PCNA-Ub assay, USP1 inhibitor + PARP inhibitor combination in xenograft and patient-derived organoids","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — RAD18 rescue experiment plus organoid validation, mechanistic link to PCNA ubiquitination","pmids":["38885312"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of USP1 with KSQ-4279 (RO7623066) shows it binds to the same cryptic hydrophobic tunnel-like site as ML323 but disrupts USP1 protein structure in subtly different ways; both inhibitors cause substantial increases in USP1 thermal stability.","method":"Cryo-EM, biochemical inhibition assay, thermal stability assay","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with biochemical validation of inhibitor binding mode","pmids":["39190802"],"is_preprint":false},{"year":2024,"finding":"USP1 deubiquitinates BRD4 in vivo and in vitro, increasing BRD4 stability; P53 indirectly represses USP1 transcription through the P21-RB1 axis, thereby promoting BRD4 degradation in liver cancer.","method":"In vivo and in vitro ubiquitination/deubiquitination assay, Co-IP, transcriptional reporter, xenograft model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro and in vivo deubiquitination assay with pathway epistasis, single lab","pmids":["38309505"],"is_preprint":false},{"year":2024,"finding":"USP1 suppresses cGAS-STING signaling by removing K27-linked oligo-ubiquitination from SAR1A; SAR1A ubiquitination is required to assemble the STING-COP-II transport complex for STING trafficking from ER to Golgi; USP1 inhibition sustains SAR1A ubiquitination and enhances STING activation and Type I IFN production.","method":"Chemical DUB screen, Co-IP, ubiquitination linkage assay (K27), STING trafficking assay, IFN reporter, in vivo radiotherapy model","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — chemical screen plus mechanistic ubiquitination linkage and trafficking assay, single lab","pmids":["39976106"],"is_preprint":false},{"year":2024,"finding":"The UAF1-USP1 complex interacts with cGAS, selectively cleaves K48-linked polyubiquitination on cGAS, and stabilizes cGAS protein expression, thereby enhancing cGAS-dependent type I IFN responses during viral infection.","method":"Co-IP, ubiquitination (K48 linkage) assay, UAF1 KO, ML323 inhibitor, in vitro and in vivo antiviral assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with K48-linkage specificity and in vivo validation, single lab","pmids":["38054892"],"is_preprint":false},{"year":2016,"finding":"Usp1 plays a role in centrosome duplication; ectopic expression of catalytically active but not the C90S inactive mutant induced centrosome amplification, while Usp1 ablation in MEFs delayed centrosome duplication; loss of ID1 suppressed Usp1-induced centrosome amplification, indicating ID1 is downstream.","method":"Ectopic expression of WT and C90S mutant Usp1, centrosome number assay, Usp1 KO MEFs, ID1 epistasis","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 — active-site mutant control plus epistasis with ID1, single lab","pmids":["26822809"],"is_preprint":false},{"year":2018,"finding":"USP1 deubiquitinates and stabilizes EZH2 in glioma cells; β-catenin/TCF4 activates USP1 transcription, and USP1-stabilized EZH2 is recruited to promoters of CDKN1B, RUNX3, and HOXA5, enhancing H3K27me3 and repressing target gene expression.","method":"Co-IP, ubiquitination assay, ChIP (EZH2 and H3K27me3), transcriptional reporter, xenograft model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus ChIP with functional transcriptional readout, single lab","pmids":["30425057"],"is_preprint":false},{"year":2011,"finding":"USP1 is phosphorylated by cyclin-dependent kinases (Cdks) in mitosis, and this phosphorylation may prevent premature APC/C(Cdh1)-mediated degradation of USP1 during normal cell cycle progression.","method":"Cell cycle analysis, Cdk phosphorylation assay, APC/C(Cdh1) substrate assay","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 3 — mechanistic follow-up paper, single lab, biochemical evidence","pmids":["22101265"],"is_preprint":false},{"year":2021,"finding":"The USP1-WDR48 complex stabilizes TAK1 by removing its polyubiquitination in vitro; loss of USP1 or WDR48 reduces phosphorylation of Smad2/3, Erk, Jnk, and p38, impairing TGF-β-mediated EMT in TNBC cells.","method":"Co-IP, in vitro ubiquitination/deubiquitination assay, phosphorylation western blot, EMT/migration assay","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 3 — in vitro deubiquitination assay with functional signaling readout, single lab","pmids":["33461373"],"is_preprint":false},{"year":2020,"finding":"USP1 deubiquitinates and stabilizes TBLR1 (transducin β-like 1 X-linked receptor 1) in hepatocellular circulating tumor cells, promoting Wnt signaling and CTC survival.","method":"Co-IP, ubiquitination assay, USP1 KO, xenograft/CTC survival assay","journal":"Frontiers in oncology","confidence":"Low","confidence_rationale":"Tier 3 — Co-IP with single functional readout, single lab","pmids":["33102219"],"is_preprint":false},{"year":2025,"finding":"USP1 can be harnessed in DUBTAC (deubiquitinase-targeting chimera) technology to stabilize target proteins; USP1-recruiting DUBTACs (e.g., MS5310 for CFTR, MS7131 for UTX) effectively stabilize their targets in a manner dependent on engagement of both USP1 and the target, demonstrating USP1 can be redirected to deubiquitinate non-native substrates.","method":"DUBTAC bifunctional molecule, protein stability assay, CFTR and UTX deubiquitination, cancer cell proliferation assay","journal":"Journal of the American Chemical Society","confidence":"Medium","confidence_rationale":"Tier 2 — chemical biology approach with orthogonal functional validation, single lab","pmids":["40252079"],"is_preprint":false},{"year":2020,"finding":"USP1 interacts with and deubiquitinates PHLPP1, stabilizing it; USP1 silencing significantly decreases the half-life of PHLPP1, which amplifies Akt1 phosphorylation, establishing a USP1-PHLPP1-Akt signaling axis in lung cancer cells.","method":"Co-IP, GST pull-down, CHX half-life assay, siRNA knockdown, phospho-Akt western blot","journal":"Journal of cancer research and clinical oncology","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus half-life assay with functional readout, single lab","pmids":["22426999"],"is_preprint":false}],"current_model":"USP1, a cysteine deubiquitinase activated by its obligate WD40 cofactor UAF1 through active-site modulation, deubiquitinates monoubiquitinated FANCD2/FANCI and PCNA at replication forks to regulate the Fanconi anemia, translesion synthesis, and homologous recombination DNA repair pathways; it also targets diverse non-DNA-repair substrates including ID1/2/3, Akt (K63-linked), TBK1, cGAS, TAZ, CHK1, and numerous oncoproteins to control differentiation, immune signaling, and tumor progression, with USP1 activity itself tightly regulated by APC/C(Cdh1)-mediated G1 degradation, Cdk-mediated mitotic phosphorylation, autocleavage/Spartan-mediated recycling from DNA, and DNA-stimulated allosteric activation through its insert L1 domain."},"narrative":{"teleology":[{"year":1998,"claim":"Establishing that USP1 is a catalytically active deubiquitinating enzyme resolved the basic question of whether this USP-family gene product possesses genuine protease activity.","evidence":"In vitro cleavage of Ub-beta-galactosidase fusion by recombinant USP1","pmids":["9806842"],"confidence":"High","gaps":["No physiological substrate identified","No cellular function assigned"]},{"year":2005,"claim":"Identifying FANCD2 as the first physiological substrate of USP1 placed it squarely in the Fanconi anemia DNA repair pathway, explaining how monoubiquitinated FANCD2 is reversed after damage resolution.","evidence":"RNAi screen, Co-IP, chromatin colocalization, chromosomal aberration assays in human cells","pmids":["15694335"],"confidence":"High","gaps":["Mechanism of substrate specificity unknown","Whether USP1 acts alone or requires cofactors unresolved"]},{"year":2009,"claim":"Mouse Usp1 knockout producing an FA-like phenotype with impaired HR and epistasis with Fancd2 confirmed USP1 as essential for proper FA pathway function in vivo, not merely redundant.","evidence":"Targeted Usp1 KO and Usp1/Fancd2 double KO in mice with crosslinker sensitivity, HR, and fertility assays","pmids":["19217432"],"confidence":"High","gaps":["Biochemical mechanism of USP1 activation not yet defined","PCNA substrate role not yet genetically validated in vivo"]},{"year":2010,"claim":"Discovery that ELG1 directs the USP1-UAF1 complex specifically to PCNA without affecting FANCD2 deubiquitination revealed how a single DUB achieves substrate selectivity through adaptor proteins.","evidence":"Co-IP, siRNA, domain mapping showing ELG1 N-terminus mediates USP1-UAF1 interaction and selectively controls PCNA-Ub levels","pmids":["20147293"],"confidence":"High","gaps":["Structural basis of ELG1-mediated substrate selection unknown","Whether other adaptors exist for additional substrates unresolved"]},{"year":2011,"claim":"Multiple discoveries in 2011 expanded USP1's roles beyond DNA repair: deubiquitination of ID1/2/3 controlling osteogenic differentiation, APC/C(Cdh1)-mediated G1 degradation coupling USP1 to cell-cycle-regulated PCNA ubiquitination, and promotion of HR by suppressing NHEJ collectively established USP1 as a multi-pathway regulator with tight temporal control.","evidence":"ID protein Co-IP/deubiquitination/differentiation assays; cell-cycle synchronization with non-degradable USP1 mutant; DT40 genetic epistasis with NHEJ-deficient cells","pmids":["21925315","21768287","21482670","22101265"],"confidence":"High","gaps":["How USP1 balances multiple substrates at different cell-cycle phases not resolved","Cdk phosphorylation sites on USP1 and their individual contributions unclear"]},{"year":2012,"claim":"Kinetic characterization showed UAF1 activates USP1 by lowering the catalytic histidine pKa, providing the first mechanistic explanation for cofactor-dependent activation, while discovery of N-end rule degradation of the autocleavage fragment explained how USP1 self-limits its activity.","evidence":"pH-dependent kinetics and solvent isotope effects for UAF1 activation; Ntaq1-mediated deamidation of autocleavage fragment with UV sensitivity phenotype","pmids":["22439892","23159736"],"confidence":"High","gaps":["No high-resolution structure of USP1-UAF1 complex yet","How autocleavage is triggered in cells unknown"]},{"year":2014,"claim":"Development of ML323 as a selective USP1-UAF1 inhibitor pharmacologically validated USP1 as essential for both the FA and TLS pathways and opened therapeutic exploration of USP1 inhibition.","evidence":"ML323 selectivity profiling, PCNA-Ub and FANCD2-Ub cellular assays, cisplatin potentiation","pmids":["24531842"],"confidence":"High","gaps":["Structural basis of ML323 inhibition unknown at this time","In vivo efficacy of USP1 inhibition not yet demonstrated"]},{"year":2018,"claim":"Demonstration that USP1 is directly stimulated by fork DNA through its DNA-binding region, and that DNA-binding-deficient USP1 fails to protect replication forks, established that USP1 functions as a DNA-activated enzyme at sites of replication stress.","evidence":"DNA-binding assays, truncation mutants lacking DNA-binding domain, replication fork protection assays in BRCA1-deficient cells","pmids":["30576655"],"confidence":"High","gaps":["Structural basis of DNA-mediated activation not resolved","Whether DNA stimulation applies equally to all substrates unclear"]},{"year":2019,"claim":"Biochemical reconstitution showed that efficient FANCD2 deubiquitination requires DNA binding by UAF1 (or RAD51AP1), unifying DNA sensing and substrate engagement into a coherent activation mechanism for the FA pathway.","evidence":"Reconstituted deubiquitination with DNA-binding mutants of UAF1 and RAD51AP1, cellular FANCD2-Ub assays","pmids":["31253762"],"confidence":"High","gaps":["Relative contributions of UAF1 vs. RAD51AP1 DNA binding in vivo not quantified"]},{"year":2021,"claim":"Crystal and cryo-EM structures of USP1-UAF1 alone and bound to monoubiquitinated FANCI-FANCD2 revealed that UAF1 drives conformational changes in FANCI-FANCD2 to present the ubiquitinated lysine, while the USP1 N-terminus confers FANCD2 specificity; insert L1 acts as an autoinhibitory element relieved by UAF1 and DNA.","evidence":"X-ray crystallography, cryo-EM, mutagenesis of UAF1-FANCI interface, insert L1/L3 deletion mutants with biochemical activity assays, N-terminal chimera experiments","pmids":["33795880","33619839","30456385"],"confidence":"High","gaps":["Full-length USP1-UAF1-DNA-PCNA complex structure not available","How insert L1 communicates with the active site at atomic level not fully resolved"]},{"year":2022,"claim":"Autocleavage-defective USP1 causes replication fork stalling resolved by Spartan-mediated removal, establishing a recycling mechanism that prevents USP1 from becoming a DNA-protein crosslink-like obstacle, while cryo-EM of ML323-bound USP1 revealed a cryptic hydrophobic tunnel as the inhibitor binding site.","evidence":"Super-resolution and single-molecule tracking of autocleavage mutant; cryo-EM at 2.5 Å of USP1±ML323","pmids":["35365626","36170365"],"confidence":"High","gaps":["Whether Spartan processes autocleavage-competent USP1 under normal conditions unclear","Dynamics of cryptic tunnel opening not characterized"]},{"year":2022,"claim":"Genome-wide CRISPR screens identified RAD18 and UBE2K as required for USP1 dependency, and PCNA K164R rescued USP1 inhibitor sensitivity, definitively establishing that the cytotoxic mechanism of USP1 loss involves aberrant accumulation of ubiquitinated PCNA.","evidence":"CRISPR-Cas9 screens, PCNA WT vs K164R rescue, DNA synthesis assays","pmids":["36228090"],"confidence":"High","gaps":["Which downstream effectors of PCNA-Ub cause cell death not fully resolved","Whether FANCD2 deubiquitination contributes to cytotoxicity in this context unclear"]},{"year":2024,"claim":"USP1 was shown to promote PRIMPOL-generated ssDNA gap accumulation expanded by MRE11/EXO1, and RAD18-dependent PCNA-Ub was confirmed as the mechanism of USP1 inhibitor synthetic lethality in BRCA1-deficient cells, completing the mechanistic link from USP1 to replication gap-driven cell death.","evidence":"ssDNA gap assays, RAD18 KD rescue, patient-derived organoid validation of USP1 inhibitor + PARP inhibitor combination","pmids":["38180818","38885312"],"confidence":"High","gaps":["Whether ssDNA gap toxicity is the sole mechanism or synergizes with fork collapse remains open","Clinical translation of USP1 inhibitor combinations awaits trial data"]},{"year":2024,"claim":"USP1 was found to suppress cGAS-STING signaling by removing K27-linked ubiquitin from SAR1A required for STING ER-to-Golgi trafficking, while separately stabilizing cGAS itself via K48-linked deubiquitination, revealing a dual and context-dependent role in innate immune regulation.","evidence":"Chemical DUB screen, K27 and K48 linkage-specific ubiquitination assays, STING trafficking, IFN reporter, in vivo viral and radiotherapy models","pmids":["39976106","38054892"],"confidence":"Medium","gaps":["How USP1 discriminates between SAR1A and cGAS under different stimuli unknown","Whether these innate immune functions require UAF1 not explicitly tested for SAR1A"]},{"year":null,"claim":"A full structural understanding of how USP1-UAF1 engages PCNA on DNA, the mechanism by which USP1 selects among its growing list of substrates in different cellular contexts, and the in vivo therapeutic window of USP1 inhibitors remain open questions.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of USP1-UAF1 bound to ubiquitinated PCNA on DNA","Substrate prioritization mechanism across DNA repair, differentiation, and immune contexts unresolved","Clinical efficacy and toxicity of USP1 inhibitors not yet established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,3,9,27,28]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,9]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[17,22]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[10,17]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,17,35]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,2,5,12,14,22,27]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[4,6,17,35,36,49,50]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,7,57]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[16,53,54]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[8,9,27]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,15,44]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[24,38,40,45]}],"complexes":["USP1-UAF1"],"partners":["UAF1","FANCD2","FANCI","PCNA","RAD51AP1","ELG1","ID1","TBK1"],"other_free_text":[]},"mechanistic_narrative":"USP1 is a cysteine deubiquitinase that serves as a central regulator of DNA damage tolerance and repair by deubiquitinating monoubiquitinated FANCD2/FANCI and PCNA at replication forks, thereby controlling the Fanconi anemia interstrand crosslink repair pathway, translesion synthesis, and homologous recombination [PMID:15694335, PMID:19217432, PMID:30576655, PMID:38885312]. USP1 requires the WD40 cofactor UAF1 (WDR48) for full catalytic activation through allosteric modulation of the catalytic histidine pKa, and substrate recognition is further specified by the USP1 N-terminus (for FANCD2) and accessory factors such as ELG1 (for PCNA) and RAD51AP1, with DNA itself serving as an allosteric activator via USP1's insert L1 domain [PMID:22439892, PMID:20147293, PMID:31253762, PMID:33619839, PMID:30456385]. USP1 activity is tightly cell-cycle-regulated through APC/C(Cdh1)-mediated G1 degradation, Cdk-dependent mitotic phosphorylation, and autocleavage coupled to Spartan-mediated removal from replication forks [PMID:21768287, PMID:22101265, PMID:35365626]. Beyond DNA repair, USP1 deubiquitinates diverse substrates including ID1/2/3 to block osteogenic differentiation, TBK1 and cGAS to modulate innate immune signaling, TAZ to regulate Hippo pathway output and Th17/Treg balance, and K63-linked ubiquitin on Akt to restrict PI3K signaling during starvation [PMID:21925315, PMID:29138248, PMID:38054892, PMID:36600049, PMID:32133736]."},"prefetch_data":{"uniprot":{"accession":"O94782","full_name":"Ubiquitin carboxyl-terminal hydrolase 1","aliases":["Deubiquitinating enzyme 1","hUBP","Ubiquitin thioesterase 1","Ubiquitin-specific-processing protease 1"],"length_aa":785,"mass_kda":88.2,"function":"Negative regulator of DNA damage repair which specifically deubiquitinates monoubiquitinated FANCD2 (PubMed:15694335). Also involved in PCNA-mediated translesion synthesis (TLS) by deubiquitinating monoubiquitinated PCNA (PubMed:16531995, PubMed:20147293). Has almost no deubiquitinating activity by itself and requires the interaction with WDR48 to have a high activity (PubMed:18082604, PubMed:26388029)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/O94782/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP1","classification":"Not Classified","n_dependent_lines":247,"n_total_lines":1208,"dependency_fraction":0.20447019867549668},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000162607","cell_line_id":"CID000804","localizations":[{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"WDR48","stoichiometry":10.0},{"gene":"KPNB1","stoichiometry":0.2},{"gene":"HIBADH","stoichiometry":0.2},{"gene":"UBE3B","stoichiometry":0.2},{"gene":"KPNA1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000804","total_profiled":1310},"omim":[{"mim_id":"620846","title":"N-TERMINAL GLUTAMINE AMIDASE 1; NTAQ1","url":"https://www.omim.org/entry/620846"},{"mim_id":"616086","title":"SprT-LIKE N-TERMINAL DOMAIN PROTEIN; SPRTN","url":"https://www.omim.org/entry/616086"},{"mim_id":"612492","title":"UBIQUITIN-SPECIFIC PROTEASE 30; USP30","url":"https://www.omim.org/entry/612492"},{"mim_id":"612167","title":"WD REPEAT-CONTAINING PROTEIN 48; WDR48","url":"https://www.omim.org/entry/612167"},{"mim_id":"610186","title":"UBIQUITIN-SPECIFIC PEPTIDASE 17-LIKE FAMILY MEMBER 2; USP17L2","url":"https://www.omim.org/entry/610186"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/USP1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O94782","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94782","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94782-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94782-F1-predicted_aligned_error_v6.png","plddt_mean":59.59},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP1","jax_strain_url":"https://www.jax.org/strain/search?query=USP1"},"sequence":{"accession":"O94782","fasta_url":"https://rest.uniprot.org/uniprotkb/O94782.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94782/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94782"}},"corpus_meta":[{"pmid":"15694335","id":"PMC_15694335","title":"The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.","date":"2005","source":"Molecular 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USP1/ UAF1 deubiquitinase complex reverse cisplatin resistance in non-small cell lung cancer cells.","date":"2011","source":"Chemistry & biology","url":"https://pubmed.ncbi.nlm.nih.gov/22118673","citation_count":192,"is_preprint":false},{"pmid":"24130053","id":"PMC_24130053","title":"Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells.","date":"2013","source":"Molecular cancer therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/24130053","citation_count":154,"is_preprint":false},{"pmid":"30576655","id":"PMC_30576655","title":"USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.","date":"2018","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/30576655","citation_count":154,"is_preprint":false},{"pmid":"23937906","id":"PMC_23937906","title":"USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy.","date":"2013","source":"Molecular 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38309505","citation_count":7,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48699,"output_tokens":13442,"usd":0.173864},"stage2":{"model":"claude-opus-4-6","input_tokens":17756,"output_tokens":3944,"usd":0.28107},"total_usd":0.454934,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"USP1 physically associates with FANCD2 and deubiquitinates monoubiquitinated FANCD2, with both proteins colocalizing on chromatin after DNA damage; RNAi inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2 and chromosomal aberrations, placing USP1 as a deubiquitinase in the Fanconi anemia pathway.\",\n      \"method\": \"RNAi library screen, Co-IP, colocalization, chromosomal aberration assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus functional KD phenotype, replicated widely across subsequent studies\",\n      \"pmids\": [\"15694335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Recombinant USP1 protein possesses genuine ubiquitin-specific protease (UBP) activity, correctly cleaving a Ub-beta-galactosidase fusion to produce free ubiquitin and beta-galactosidase, identifying USP1 as an active deubiquitinating enzyme.\",\n      \"method\": \"In vitro ubiquitin cleavage assay with recombinant protein\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical reconstitution of enzymatic activity\",\n      \"pmids\": [\"9806842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Targeted deletion of mouse Usp1 results in elevated monoubiquitinated Fancd2 in chromatin, impaired Fancd2 foci assembly, defective homologous recombination repair, and an FA-like phenotype (crosslinker hypersensitivity, male infertility, perinatal lethality); double KO of Usp1 and Fancd2 yields a more severe phenotype, placing Usp1 downstream of Fancd2 monoubiquitination in the FA pathway.\",\n      \"method\": \"Targeted gene knockout in mice, epistasis (double KO), chromatin fractionation, HR assay\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with double-KO, multiple orthogonal cellular phenotype assays\",\n      \"pmids\": [\"19217432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP1 binds, deubiquitinates, and thereby stabilizes the ID1, ID2, and ID3 inhibitor-of-differentiation proteins; USP1 knockdown in osteosarcoma cells leads to ID protein destabilization, cell-cycle arrest, and osteogenic differentiation, while ectopic USP1 in mesenchymal stem cells stabilizes IDs and inhibits osteoblastic differentiation.\",\n      \"method\": \"Co-IP, ubiquitination assay, siRNA knockdown, ectopic expression, in vivo mouse model\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including Co-IP, in vitro deubiquitination, and in vivo validation\",\n      \"pmids\": [\"21925315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human ELG1 interacts with the USP1-UAF1 complex and specifically directs USP1-UAF1 to deubiquitinate PCNA; ELG1 knockdown increases PCNA monoubiquitination without affecting FANCD2 ubiquitination, and a conserved N-terminal domain of ELG1 mediates USP1-UAF1 interaction.\",\n      \"method\": \"Co-IP, siRNA knockdown, domain mapping, ubiquitination assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including Co-IP, domain mapping, and selective substrate phenotype\",\n      \"pmids\": [\"20147293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP1 and its binding partner UAF1 form a complex that promotes homologous recombination (HR)-mediated double-strand break repair; loss of UAF1 in chicken DT40 cells causes sensitivity to camptothecin and PARP inhibitors that is rescued by disrupting NHEJ, indicating USP1/UAF1 promotes HR by suppressing NHEJ.\",\n      \"method\": \"Genetic KO in DT40 cells, epistasis with NHEJ-deficient cells, drug sensitivity assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple drug sensitivity assays and pathway rescue\",\n      \"pmids\": [\"21482670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP1 is degraded in G1 via APC/C(Cdh1)-mediated ubiquitination; low USP1 levels in G1 are required for PCNA monoubiquitination in response to UV damage before DNA replication, as a non-degradable USP1 mutant inhibits PCNA monoubiquitination and likely impairs translesion synthesis polymerase recruitment.\",\n      \"method\": \"Cell cycle synchronization, APC/C(Cdh1) substrate assay, USP1 mutant expression, PCNA ubiquitination assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-cycle-specific degradation confirmed with non-degradable mutant and functional PCNA ubiquitination readout\",\n      \"pmids\": [\"21768287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP1 depletion stimulates DDB1-dependent degradation of phosphorylated CHK1; USP1 maintains both total and phosphorylated CHK1 levels in response to genotoxic stress, partly in a monoubiquitinated FANCD2-dependent manner, establishing a feedback circuit where activated CHK1 upregulates FANCD2 monoubiquitination, which then promotes USP1-mediated CHK1 degradation.\",\n      \"method\": \"siRNA knockdown, western blot, Co-IP, genotoxic stress assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — single lab, multiple methods but complex feedback circuit partially inferred\",\n      \"pmids\": [\"21389083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The C-terminal autocleavage fragment of USP1 (Gln-Usp1(Ct)) is a physiological substrate of the Arg/N-end rule pathway, targeted for degradation through deamidation of its N-terminal glutamine by Ntaq1; the autocleaved fragments remain associated with UAF1 and retain deubiquitylase activity until Gln-Usp1(Ct) is selectively destroyed, and stabilization of this fragment decreases PCNA monoubiquitylation and increases UV sensitivity.\",\n      \"method\": \"Biochemical reconstitution, N-end rule substrate identification, mutant analysis, cellular UV sensitivity assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstitution of N-end rule degradation plus functional cellular validation with UV sensitivity\",\n      \"pmids\": [\"23159736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"USP1 activity is stimulated through formation of a tight complex with the WD40 repeat protein UAF1; kinetic analyses show UAF1 activates USP1 by lowering the pKa of the catalytic histidine (by 0.43 pH units), facilitating general base catalysis at neutral pH without altering ubiquitin binding affinity; the catalytic triad is C90, H593, and D751.\",\n      \"method\": \"Kinetic analysis (pH-dependent inactivation, solvent isotope effects, proton inventory), in vitro biochemical assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — detailed mechanistic enzymology with kinetic parameters and active-site characterization\",\n      \"pmids\": [\"22439892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"USP1 contains two nuclear localization signals (NLSs) that mediate nuclear import of the USP1/UAF1 complex; USP1 and UAF1 form a complex in the cytoplasm that translocates to the nucleus via USP1 NLSs, and a 100 amino acid motif in USP1 mediates UAF1 binding.\",\n      \"method\": \"Nuclear relocation assay, deletion mutagenesis, cellular localization\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization with functional consequence mapped by mutagenesis, single lab\",\n      \"pmids\": [\"22701671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CAPNS1 (calpain regulatory subunit) is required for USP1 stability; calpain stabilizes USP1 by activating Cdk5, which inhibits Cdh1 and consequently prevents APC/C(Cdh1)-mediated USP1 degradation in G1; loss of CAPNS1 increases PCNA monoubiquitination and polymerase-η loading.\",\n      \"method\": \"siRNA knockdown, western blot, chromatin fractionation, cell-cycle analysis, Cdk5/p25 rescue\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — genetic manipulation with rescue experiment, single lab\",\n      \"pmids\": [\"23589330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The selective USP1-UAF1 inhibitor ML323 inhibits the USP1-UAF1 complex with excellent selectivity; pharmacological inhibition reveals that USP1-UAF1 deubiquitination is required for the DNA translesion synthesis and Fanconi anemia pathways in response to UV- and cisplatin-induced DNA damage, and ML323 potentiates cisplatin cytotoxicity.\",\n      \"method\": \"Chemical inhibitor (ML323), cell-based PCNA and FANCD2 ubiquitination assays, selectivity profiling\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — highly selective chemical probe with orthogonal substrate readouts across multiple DNA damage pathways\",\n      \"pmids\": [\"24531842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP1 phosphorylation at S313 is not critical for PCNA deubiquitination or UAF1 binding; amino acid motif 420-520 in USP1 is necessary and sufficient for UAF1 binding (mapping to the Fingers subdomain); USP1 autocleavage occurs in cis at the G670/G671 diglycine motif and can be reduced by cancer-associated mutation L669P.\",\n      \"method\": \"Mutagenesis (S313A, S313D, deletion mutants, GG/AA, cancer-associated mutants), cell-based UAF1 binding and PCNA deubiquitination assays\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic structure-function mutagenesis, single lab\",\n      \"pmids\": [\"25744535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The USP1-UAF1 complex interacts with RAD51AP1 (a RAD51-interacting protein) via UAF1; USP1-UAF1 stabilizes RAD51AP1 and promotes HR repair independently of FANCD2 deubiquitination; cells expressing UAF1-interaction-deficient RAD51AP1 show increased chromosomal aberrations and persistent RAD51 foci after DNA damage.\",\n      \"method\": \"Proteomics, Co-IP, domain mapping, HR assay, RAD51 foci, chromosomal aberration assay\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS interactome plus functional epistasis, single lab\",\n      \"pmids\": [\"27463890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PDGF signaling upregulates USP1 transcription through E2F transcription factors that directly bind and activate the Usp1 promoter; USP1 then stabilizes ID2 protein, which is required for proneural glioma cell survival.\",\n      \"method\": \"Mouse model, comparative transcriptomics, ChIP (E2F binding to Usp1 promoter), USP1 KO, ID2 protein stability assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus genetic ablation with in vivo tumor model, single lab\",\n      \"pmids\": [\"26951930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The USP1-UAF1 complex interacts with TBK1, removes its K48-linked polyubiquitination, and prevents TBK1 proteasomal degradation, thereby stabilizing TBK1 and enhancing TLR3/4- and RIG-I-induced IRF3 activation and IFN-β secretion during antiviral responses.\",\n      \"method\": \"Co-IP, ubiquitination assay, KD/KO, IFN-β reporter assay, in vivo viral infection model\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional deubiquitination readout with in vivo validation, single lab\",\n      \"pmids\": [\"29138248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP1 binds to and is stimulated by fork DNA; a truncated USP1 lacking its DNA-binding region is not stimulated by DNA, fails to localize to replication forks, and cannot protect replication forks; persistence of monoubiquitinated PCNA at the replication fork is the mechanism of cell death when USP1 is absent in BRCA1-deficient cells.\",\n      \"method\": \"DNA-binding assay, truncation mutants, replication fork protection assay, PCNA-Ub localization, cell viability\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple mechanistic assays with defined truncation mutants and specific cellular phenotype in BRCA1-deficient cells\",\n      \"pmids\": [\"30576655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP1 deubiquitinates and stabilizes KPNA2 (importin subunit alpha-2) protein; USP1-mediated KPNA2 stabilization is the downstream event crucial for USP1's pro-metastatic function in breast cancer cells.\",\n      \"method\": \"Co-IP, ubiquitination assay, siRNA/inhibitor-based KD, metastasis assay in mice\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP and deubiquitination assay with in vivo metastasis readout, single lab\",\n      \"pmids\": [\"30531833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP1 interacts with and deubiquitinates ULK1 via K63-linked ubiquitin chains; USP1 depletion or inhibition causes ULK1 sequestration to an insoluble/aggresome fraction and inhibits canonical autophagy while promoting an alternative lysosomal-mediated degradation route.\",\n      \"method\": \"Co-IP, ubiquitination linkage assay, cell fractionation, immunofluorescence, autophagic flux assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with K63 linkage specificity and subcellular localization with functional consequence, single lab\",\n      \"pmids\": [\"30335599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP1 positively regulates protein stability of ID1 and CHK1 in glioblastoma stem-like cells, and USP1 inhibition attenuates clonogenic growth and enhances radiosensitivity.\",\n      \"method\": \"shRNA knockdown, pimozide inhibitor, protein stability assay, clonogenic assay, in vivo mouse model\",\n      \"journal\": \"Neuro-oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — KD with protein stability readout, single lab\",\n      \"pmids\": [\"26032834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Upon platinum treatment, USP1 is phosphorylated by ATM and ATR, binds to Snail, deubiquitinates and stabilizes Snail expression, conferring platinum resistance and promoting tumor dissemination; USP1 KO or inhibition increased platinum sensitivity in a Snail-dependent manner.\",\n      \"method\": \"Co-IP, in vivo ubiquitination assay, ATM/ATR phosphorylation assay, rescue experiments with Snail overexpression, in vivo mouse model\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical methods with in vivo validation, single lab\",\n      \"pmids\": [\"31086816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Efficient FANCD2 deubiquitination by the USP1-UAF1 complex requires DNA and is dependent on DNA binding by UAF1; RAD51AP1, via its own DNA-binding activity, can substitute for UAF1 DNA binding in FANCD2 deubiquitination in reconstituted biochemical systems and in cells.\",\n      \"method\": \"Reconstituted biochemical deubiquitination assay, DNA-binding mutants of UAF1 and RAD51AP1, cellular FANCD2 deubiquitination assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted biochemical system with mutagenesis and cellular validation\",\n      \"pmids\": [\"31253762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TonEBP sequentially recruits E3 ubiquitin ligase SHPRH and then USP1 to DNA damage sites, coordinating PCNA polyubiquitination dynamics; the Rel-homology domain of TonEBP is essential for interaction with both SHPRH and USP1.\",\n      \"method\": \"Co-IP, DNA damage recruitment assay, domain mapping, PCNA polyubiquitination assay\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and recruitment assay, single lab\",\n      \"pmids\": [\"31376680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP1 removes K63-linked polyubiquitin chains on Akt to restrict PI3K-Akt-FoxO signaling in mouse muscle during prolonged starvation; Dab2 is essential for recruiting Akt to a USP1-TSC1-PHLPP1 complex for deubiquitination, and USP1 depletion increases Akt ubiquitination and glucose uptake during fasting.\",\n      \"method\": \"DUB screening platform, Co-IP, mass spectrometry, ubiquitination (K63-linkage) assay, in vivo mouse muscle USP1 depletion\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — DUB screen plus Co-IP/MS complex identification and in vivo validation, single lab\",\n      \"pmids\": [\"32133736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP1 interacts with and deubiquitinates ERα (estrogen receptor alpha), inhibiting K48-linked poly-ubiquitination and thereby stabilizing ERα protein in breast cancer cells.\",\n      \"method\": \"DUB siRNA library screen, Co-IP, ubiquitination assay\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — DUB screen plus Co-IP with ubiquitination assay, single lab\",\n      \"pmids\": [\"33123289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP1 maintains USP1 protein stability in part by limiting TSC1 levels to sustain mTOR-mediated basal protein synthesis that maintains USP1's own levels, creating an auto-regulatory loop.\",\n      \"method\": \"Co-IP, siRNA knockdown, protein stability assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — supporting biochemical data within a broader study, single lab\",\n      \"pmids\": [\"32133736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The crystal structure of human USP1-UAF1 with and without ubiquitin, and a cryo-EM structure of USP1-UAF1 bound to monoubiquitinated FANCI-FANCD2, reveal that UAF1 drives conformational changes in FANCI-FANCD2 for deubiquitination; an extensive UAF1-FANCI interface (confirmed by mutagenesis) explains why both UAF1 and FANCI are required despite neither being directly catalytic.\",\n      \"method\": \"X-ray crystallography, cryo-EM, mutagenesis, biochemical deubiquitination assay\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure + cryo-EM + mutagenesis + biochemical validation in a single study\",\n      \"pmids\": [\"33795880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The N-terminus of USP1 contains a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2; the N-terminus is not required for PCNA or FANCI deubiquitination, and the N-terminus alone is sufficient to confer FANCD2 specificity on a more promiscuous USP.\",\n      \"method\": \"Reconstituted deubiquitination assay with purified monoubiquitinated substrates, N-terminal deletion/chimera mutants\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical reconstitution with defined substrates and domain-swap/chimera experiments\",\n      \"pmids\": [\"30456385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Insert L1 of USP1 limits intrinsic USP1 activity and is required for both UAF1-mediated allosteric activation and substrate-dependent (DNA and PCNA) activity enhancement independently of UAF1; insert L3 also contributes to limiting intrinsic activity.\",\n      \"method\": \"Biochemical activity assays with insert deletion mutants, UAF1 activation assays, DNA/PCNA stimulation assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with multiple mutants defining allosteric mechanism\",\n      \"pmids\": [\"33619839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GRP75 forms a complex with USP1 and SIX1, recruiting USP1 to inhibit K48-linked polyubiquitination of SIX1; GRP75's C-terminus (433-679 aa) peptide-binding domain is required for GRP75-USP1-SIX1 complex formation.\",\n      \"method\": \"Co-IP, domain mapping, ubiquitination assay, xenograft model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with domain mapping and functional readout, single lab\",\n      \"pmids\": [\"34079090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP1 interacts with and deubiquitinates BCAT2 at K229; BCAA increases USP1 protein levels via the GCN2-eIF2α pathway, and USP1-mediated BCAT2 stabilization promotes PDAC development.\",\n      \"method\": \"DUB library screen, Co-IP, in vitro deubiquitination assay, K229 site mapping, GCN2-eIF2α pathway analysis\",\n      \"journal\": \"National science review\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — DUB screen plus in vitro deubiquitination with site identification, single lab\",\n      \"pmids\": [\"35663242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP1 interacts with and deubiquitinates RPS16, removing K48-linked ubiquitin chains to prevent proteasomal degradation; catalytically inactive USP1-C90A mutant cannot stabilize RPS16, confirming USP1 enzymatic activity is required.\",\n      \"method\": \"Mass spectrometry, Co-IP, western blot, C90A mutant, ubiquitination assay, xenograft model\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with active-site mutant control and in vivo validation, single lab\",\n      \"pmids\": [\"34154657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP1 deubiquitinates Aurora B to prevent its degradation, mediating glucocorticoid resistance in T-ALL; ALKBH5-mediated m6A demethylation increases USP1 mRNA stability, thereby elevating USP1 and Aurora B levels.\",\n      \"method\": \"Co-IP, deubiquitination assay, siRNA knockdown, rescue experiments, in vivo mouse model\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus functional rescue, single lab\",\n      \"pmids\": [\"34169564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM at 2.5-Å resolution of USP1 with and without ML323 reveals that ML323 binds to a cryptic hydrophobic site within USP1, disrupts part of the hydrophobic core, and causes conformational changes in secondary structure that lead to subtle rearrangements in the active site underlying inhibition.\",\n      \"method\": \"Cryo-EM (2.5 Å resolution), structure-based mechanistic analysis\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structure with mechanistic interpretation of inhibitor binding mode\",\n      \"pmids\": [\"36170365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cells harboring an autocleavage-defective USP1 mutant experience more replication fork-stalling and premature fork termination; USP1 molecules are removed from active DNA synthesis sites by the DNA-dependent metalloprotease Spartan to prevent 'USP1-trapping' lesions; autocleavage enables USP1 recycling from DNA.\",\n      \"method\": \"Autocleavage-defective mutant, super-resolution microscopy, live-cell single-molecule tracking, replication fork assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live-cell single-molecule imaging plus replication assay with defined mutant and Spartan genetic interaction\",\n      \"pmids\": [\"35365626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP1 inhibition leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage; CRISPR screens identify RAD18 and UBE2K (promoting PCNA mono- and polyubiquitination) as required mediators of USP1 dependency; ectopic expression of WT but not K164R PCNA reverses USP1 inhibitor sensitivity, placing USP1 dependency on aberrant processing of ubiquitinated PCNA.\",\n      \"method\": \"CRISPR-Cas9 genome-wide screens, PCNA ubiquitination assay, ectopic PCNA expression (WT vs K164R mutant), DNA synthesis assay\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide CRISPR screen validated by PCNA mutant rescue experiment\",\n      \"pmids\": [\"36228090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP1 interacts with and deubiquitinates MAST1, preventing its K48-linked polyubiquitination and extending its half-life; USP1-stabilized MAST1 activates MEK1, driving cisplatin resistance.\",\n      \"method\": \"CRISPR/Cas9-based genome-wide dual screen, Co-IP, domain mapping, in vitro deubiquitination assay, half-life analysis, xenograft model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR screen plus in vitro deubiquitination assay with in vivo validation, single lab\",\n      \"pmids\": [\"35966591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP1 interacts with and deubiquitinates TAZ via the Hippo pathway in osteosarcoma; USP1 inhibition by ML323 destabilizes TAZ, reducing nuclear accumulation and expression of Hippo downstream genes, suppressing OS growth and metastasis in vitro and in vivo.\",\n      \"method\": \"Co-IP, ubiquitination assay, in vitro and in vivo tumor models\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus functional readout, single lab\",\n      \"pmids\": [\"35637948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP1 interacts with and deubiquitinates MAX, maintaining its stability; USP1-stabilized MAX promotes MYC target gene transcription, contributing to chemotherapy resistance in DLBCL.\",\n      \"method\": \"Co-IP, ubiquitination assay, shRNA/pimozide inhibition, MYC target gene expression, xenograft and PDX models\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with functional rescue and in vivo validation, single lab\",\n      \"pmids\": [\"36352191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP1 deubiquitinates and stabilizes TAZ (WWTR1) in CD4+ T cells, enhancing RORγt activity and Th17 differentiation while promoting Foxp3 proteasomal degradation to suppress Treg differentiation; ML323 inhibition of USP1 shifts this balance toward Treg differentiation in vitro and in vivo.\",\n      \"method\": \"siRNA screen, Co-IP, ubiquitination assay, Th17/Treg differentiation assays, in vivo inflammatory model\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — DUB screen plus Co-IP and in vivo validation, single lab\",\n      \"pmids\": [\"36600049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The USP1-UAF1 complex interacts with and stabilizes METTL3 by removing K48-linked ubiquitination after spinal cord injury, and this stabilization promotes reactive astrogliosis through METTL3-mediated m6A methylation of YAP1 mRNA.\",\n      \"method\": \"Co-IP, ubiquitination assay, METTL3 conditional KO, m6A-seq, RNA stability assay, in vivo SCI model\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with K48-linkage specificity and in vivo rescue, single lab\",\n      \"pmids\": [\"36653190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP1 interacts with and deubiquitinates PARP1, removing the ubiquitin chain at K197 to prevent its proteasomal degradation; GCN5 acetyltransferase acetylates USP1 at K130, enhancing the USP1-PARP1 interaction and further increasing PARP1 stabilization.\",\n      \"method\": \"Proteomics, ubiquitylome analysis, Co-IP, GST pull-down, immunofluorescence, deletion mutant mapping, acetyltransferase assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomics-guided Co-IP plus ubiquitylome with site mapping and PTM writer identification, single lab\",\n      \"pmids\": [\"37821462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP1 interacts with PLK1 and deubiquitinates it; USP1-stabilized PLK1 promotes LDHA expression and aerobic glycolysis in T-ALL cells.\",\n      \"method\": \"Co-IP, deubiquitination assay, siRNA knockdown, rescue experiments with LDHA\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with functional rescue, single lab\",\n      \"pmids\": [\"36912760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP1 interacts with and deubiquitinates C/EBPβ, increasing its protein stability; catalytically inactive USP1 has no effect on C/EBPβ stability; USP1-mediated C/EBPβ stabilization promotes adipogenesis and lipid accumulation.\",\n      \"method\": \"Co-IP, ubiquitination assay, USP1 C90A catalytic mutant, adipogenic differentiation assay, HFD mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with catalytic mutant control and in vivo HFD validation, single lab\",\n      \"pmids\": [\"38012162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP1 interacts with the WW domain of TAZ in HCC and stabilizes it by suppressing K11-linked polyubiquitination; a DUB siRNA screen identified USP1 as a critical regulator of Hippo/TAZ signaling activity.\",\n      \"method\": \"DUB siRNA screen, Co-IP, ubiquitination linkage assay, RNA-seq, xenograft model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — DUB screen plus Co-IP with specific K11-linkage identification, single lab\",\n      \"pmids\": [\"37041150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Human cytomegalovirus UL138 interacts with the UAF1-USP1 complex; this interaction activates STAT1 phosphorylation (pSTAT1), and USP1 inhibition disrupts HCMV latency establishment, increasing viral replication.\",\n      \"method\": \"Co-IP, pSTAT1 assay, USP1 inhibitor (ML323), viral replication assay, ChIP of pSTAT1 on viral genome\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with functional inhibitor readout in viral infection model, single lab\",\n      \"pmids\": [\"37289831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP1 inhibitor ML323 stabilizes NLRP3 and AIM2 by deubiquitination in thyroid follicular cells, and USP1 modulates NLRP3 transcription by facilitating p65 nuclear transport; USP1 knockdown reduces pyroptosis markers in vitro and ameliorates HT in vivo.\",\n      \"method\": \"Co-IP, ubiquitination assay, KD/inhibitor, pyroptosis assay, p65 nuclear translocation assay, in vivo HT mouse model\",\n      \"journal\": \"Molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus multiple functional assays, single lab\",\n      \"pmids\": [\"39134949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP1 deubiquitinates PARP1 by removing K63-linked polyubiquitination, controlling PARP1 chromatin trapping and PARylation activity, thereby regulating sensitivity to PARP inhibitors; combined USP1/PARP1 blockade enhances replicative stress and cell death.\",\n      \"method\": \"Co-IP, K63-ubiquitination assay, PARP trapping assay, PARylation assay, cell death assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with K63-linkage specificity and multiple functional readouts (trapping, PARylation), single lab\",\n      \"pmids\": [\"39536107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP1 promotes PRIMPOL-generated ssDNA gap accumulation and their bidirectional expansion by MRE11 and EXO1 nucleases during replication stress; this activity is linked to USP1's role in PCNA deubiquitination, as PCNA ubiquitination prevents gap accumulation.\",\n      \"method\": \"siRNA depletion, ssDNA gap assay, nuclease inhibitor assays, PCNA ubiquitination assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays with defined mechanistic link to PCNA deubiquitination, single lab\",\n      \"pmids\": [\"38180818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP1 inhibition promotes ssDNA gap accumulation at replication forks via RAD18-dependent PCNA monoubiquitination; knockdown of RAD18 causes USP1 inhibitor resistance and suppresses ssDNA gap formation, establishing this as the mechanism of USP1 inhibitor-induced synthetic lethality in BRCA1-deficient cells.\",\n      \"method\": \"ssDNA gap assay, RAD18 KD rescue, PCNA-Ub assay, USP1 inhibitor + PARP inhibitor combination in xenograft and patient-derived organoids\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — RAD18 rescue experiment plus organoid validation, mechanistic link to PCNA ubiquitination\",\n      \"pmids\": [\"38885312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of USP1 with KSQ-4279 (RO7623066) shows it binds to the same cryptic hydrophobic tunnel-like site as ML323 but disrupts USP1 protein structure in subtly different ways; both inhibitors cause substantial increases in USP1 thermal stability.\",\n      \"method\": \"Cryo-EM, biochemical inhibition assay, thermal stability assay\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with biochemical validation of inhibitor binding mode\",\n      \"pmids\": [\"39190802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP1 deubiquitinates BRD4 in vivo and in vitro, increasing BRD4 stability; P53 indirectly represses USP1 transcription through the P21-RB1 axis, thereby promoting BRD4 degradation in liver cancer.\",\n      \"method\": \"In vivo and in vitro ubiquitination/deubiquitination assay, Co-IP, transcriptional reporter, xenograft model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo deubiquitination assay with pathway epistasis, single lab\",\n      \"pmids\": [\"38309505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP1 suppresses cGAS-STING signaling by removing K27-linked oligo-ubiquitination from SAR1A; SAR1A ubiquitination is required to assemble the STING-COP-II transport complex for STING trafficking from ER to Golgi; USP1 inhibition sustains SAR1A ubiquitination and enhances STING activation and Type I IFN production.\",\n      \"method\": \"Chemical DUB screen, Co-IP, ubiquitination linkage assay (K27), STING trafficking assay, IFN reporter, in vivo radiotherapy model\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — chemical screen plus mechanistic ubiquitination linkage and trafficking assay, single lab\",\n      \"pmids\": [\"39976106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The UAF1-USP1 complex interacts with cGAS, selectively cleaves K48-linked polyubiquitination on cGAS, and stabilizes cGAS protein expression, thereby enhancing cGAS-dependent type I IFN responses during viral infection.\",\n      \"method\": \"Co-IP, ubiquitination (K48 linkage) assay, UAF1 KO, ML323 inhibitor, in vitro and in vivo antiviral assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with K48-linkage specificity and in vivo validation, single lab\",\n      \"pmids\": [\"38054892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Usp1 plays a role in centrosome duplication; ectopic expression of catalytically active but not the C90S inactive mutant induced centrosome amplification, while Usp1 ablation in MEFs delayed centrosome duplication; loss of ID1 suppressed Usp1-induced centrosome amplification, indicating ID1 is downstream.\",\n      \"method\": \"Ectopic expression of WT and C90S mutant Usp1, centrosome number assay, Usp1 KO MEFs, ID1 epistasis\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — active-site mutant control plus epistasis with ID1, single lab\",\n      \"pmids\": [\"26822809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP1 deubiquitinates and stabilizes EZH2 in glioma cells; β-catenin/TCF4 activates USP1 transcription, and USP1-stabilized EZH2 is recruited to promoters of CDKN1B, RUNX3, and HOXA5, enhancing H3K27me3 and repressing target gene expression.\",\n      \"method\": \"Co-IP, ubiquitination assay, ChIP (EZH2 and H3K27me3), transcriptional reporter, xenograft model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus ChIP with functional transcriptional readout, single lab\",\n      \"pmids\": [\"30425057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP1 is phosphorylated by cyclin-dependent kinases (Cdks) in mitosis, and this phosphorylation may prevent premature APC/C(Cdh1)-mediated degradation of USP1 during normal cell cycle progression.\",\n      \"method\": \"Cell cycle analysis, Cdk phosphorylation assay, APC/C(Cdh1) substrate assay\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — mechanistic follow-up paper, single lab, biochemical evidence\",\n      \"pmids\": [\"22101265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The USP1-WDR48 complex stabilizes TAK1 by removing its polyubiquitination in vitro; loss of USP1 or WDR48 reduces phosphorylation of Smad2/3, Erk, Jnk, and p38, impairing TGF-β-mediated EMT in TNBC cells.\",\n      \"method\": \"Co-IP, in vitro ubiquitination/deubiquitination assay, phosphorylation western blot, EMT/migration assay\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — in vitro deubiquitination assay with functional signaling readout, single lab\",\n      \"pmids\": [\"33461373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP1 deubiquitinates and stabilizes TBLR1 (transducin β-like 1 X-linked receptor 1) in hepatocellular circulating tumor cells, promoting Wnt signaling and CTC survival.\",\n      \"method\": \"Co-IP, ubiquitination assay, USP1 KO, xenograft/CTC survival assay\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with single functional readout, single lab\",\n      \"pmids\": [\"33102219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP1 can be harnessed in DUBTAC (deubiquitinase-targeting chimera) technology to stabilize target proteins; USP1-recruiting DUBTACs (e.g., MS5310 for CFTR, MS7131 for UTX) effectively stabilize their targets in a manner dependent on engagement of both USP1 and the target, demonstrating USP1 can be redirected to deubiquitinate non-native substrates.\",\n      \"method\": \"DUBTAC bifunctional molecule, protein stability assay, CFTR and UTX deubiquitination, cancer cell proliferation assay\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — chemical biology approach with orthogonal functional validation, single lab\",\n      \"pmids\": [\"40252079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP1 interacts with and deubiquitinates PHLPP1, stabilizing it; USP1 silencing significantly decreases the half-life of PHLPP1, which amplifies Akt1 phosphorylation, establishing a USP1-PHLPP1-Akt signaling axis in lung cancer cells.\",\n      \"method\": \"Co-IP, GST pull-down, CHX half-life assay, siRNA knockdown, phospho-Akt western blot\",\n      \"journal\": \"Journal of cancer research and clinical oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus half-life assay with functional readout, single lab\",\n      \"pmids\": [\"22426999\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP1, a cysteine deubiquitinase activated by its obligate WD40 cofactor UAF1 through active-site modulation, deubiquitinates monoubiquitinated FANCD2/FANCI and PCNA at replication forks to regulate the Fanconi anemia, translesion synthesis, and homologous recombination DNA repair pathways; it also targets diverse non-DNA-repair substrates including ID1/2/3, Akt (K63-linked), TBK1, cGAS, TAZ, CHK1, and numerous oncoproteins to control differentiation, immune signaling, and tumor progression, with USP1 activity itself tightly regulated by APC/C(Cdh1)-mediated G1 degradation, Cdk-mediated mitotic phosphorylation, autocleavage/Spartan-mediated recycling from DNA, and DNA-stimulated allosteric activation through its insert L1 domain.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"USP1 is a cysteine deubiquitinase that serves as a central regulator of DNA damage tolerance and repair by deubiquitinating monoubiquitinated FANCD2/FANCI and PCNA at replication forks, thereby controlling the Fanconi anemia interstrand crosslink repair pathway, translesion synthesis, and homologous recombination [PMID:15694335, PMID:19217432, PMID:30576655, PMID:38885312]. USP1 requires the WD40 cofactor UAF1 (WDR48) for full catalytic activation through allosteric modulation of the catalytic histidine pKa, and substrate recognition is further specified by the USP1 N-terminus (for FANCD2) and accessory factors such as ELG1 (for PCNA) and RAD51AP1, with DNA itself serving as an allosteric activator via USP1's insert L1 domain [PMID:22439892, PMID:20147293, PMID:31253762, PMID:33619839, PMID:30456385]. USP1 activity is tightly cell-cycle-regulated through APC/C(Cdh1)-mediated G1 degradation, Cdk-dependent mitotic phosphorylation, and autocleavage coupled to Spartan-mediated removal from replication forks [PMID:21768287, PMID:22101265, PMID:35365626]. Beyond DNA repair, USP1 deubiquitinates diverse substrates including ID1/2/3 to block osteogenic differentiation, TBK1 and cGAS to modulate innate immune signaling, TAZ to regulate Hippo pathway output and Th17/Treg balance, and K63-linked ubiquitin on Akt to restrict PI3K signaling during starvation [PMID:21925315, PMID:29138248, PMID:38054892, PMID:36600049, PMID:32133736].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that USP1 is a catalytically active deubiquitinating enzyme resolved the basic question of whether this USP-family gene product possesses genuine protease activity.\",\n      \"evidence\": \"In vitro cleavage of Ub-beta-galactosidase fusion by recombinant USP1\",\n      \"pmids\": [\"9806842\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrate identified\", \"No cellular function assigned\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying FANCD2 as the first physiological substrate of USP1 placed it squarely in the Fanconi anemia DNA repair pathway, explaining how monoubiquitinated FANCD2 is reversed after damage resolution.\",\n      \"evidence\": \"RNAi screen, Co-IP, chromatin colocalization, chromosomal aberration assays in human cells\",\n      \"pmids\": [\"15694335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of substrate specificity unknown\", \"Whether USP1 acts alone or requires cofactors unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Mouse Usp1 knockout producing an FA-like phenotype with impaired HR and epistasis with Fancd2 confirmed USP1 as essential for proper FA pathway function in vivo, not merely redundant.\",\n      \"evidence\": \"Targeted Usp1 KO and Usp1/Fancd2 double KO in mice with crosslinker sensitivity, HR, and fertility assays\",\n      \"pmids\": [\"19217432\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism of USP1 activation not yet defined\", \"PCNA substrate role not yet genetically validated in vivo\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovery that ELG1 directs the USP1-UAF1 complex specifically to PCNA without affecting FANCD2 deubiquitination revealed how a single DUB achieves substrate selectivity through adaptor proteins.\",\n      \"evidence\": \"Co-IP, siRNA, domain mapping showing ELG1 N-terminus mediates USP1-UAF1 interaction and selectively controls PCNA-Ub levels\",\n      \"pmids\": [\"20147293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of ELG1-mediated substrate selection unknown\", \"Whether other adaptors exist for additional substrates unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Multiple discoveries in 2011 expanded USP1's roles beyond DNA repair: deubiquitination of ID1/2/3 controlling osteogenic differentiation, APC/C(Cdh1)-mediated G1 degradation coupling USP1 to cell-cycle-regulated PCNA ubiquitination, and promotion of HR by suppressing NHEJ collectively established USP1 as a multi-pathway regulator with tight temporal control.\",\n      \"evidence\": \"ID protein Co-IP/deubiquitination/differentiation assays; cell-cycle synchronization with non-degradable USP1 mutant; DT40 genetic epistasis with NHEJ-deficient cells\",\n      \"pmids\": [\"21925315\", \"21768287\", \"21482670\", \"22101265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How USP1 balances multiple substrates at different cell-cycle phases not resolved\", \"Cdk phosphorylation sites on USP1 and their individual contributions unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Kinetic characterization showed UAF1 activates USP1 by lowering the catalytic histidine pKa, providing the first mechanistic explanation for cofactor-dependent activation, while discovery of N-end rule degradation of the autocleavage fragment explained how USP1 self-limits its activity.\",\n      \"evidence\": \"pH-dependent kinetics and solvent isotope effects for UAF1 activation; Ntaq1-mediated deamidation of autocleavage fragment with UV sensitivity phenotype\",\n      \"pmids\": [\"22439892\", \"23159736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of USP1-UAF1 complex yet\", \"How autocleavage is triggered in cells unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Development of ML323 as a selective USP1-UAF1 inhibitor pharmacologically validated USP1 as essential for both the FA and TLS pathways and opened therapeutic exploration of USP1 inhibition.\",\n      \"evidence\": \"ML323 selectivity profiling, PCNA-Ub and FANCD2-Ub cellular assays, cisplatin potentiation\",\n      \"pmids\": [\"24531842\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of ML323 inhibition unknown at this time\", \"In vivo efficacy of USP1 inhibition not yet demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstration that USP1 is directly stimulated by fork DNA through its DNA-binding region, and that DNA-binding-deficient USP1 fails to protect replication forks, established that USP1 functions as a DNA-activated enzyme at sites of replication stress.\",\n      \"evidence\": \"DNA-binding assays, truncation mutants lacking DNA-binding domain, replication fork protection assays in BRCA1-deficient cells\",\n      \"pmids\": [\"30576655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of DNA-mediated activation not resolved\", \"Whether DNA stimulation applies equally to all substrates unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Biochemical reconstitution showed that efficient FANCD2 deubiquitination requires DNA binding by UAF1 (or RAD51AP1), unifying DNA sensing and substrate engagement into a coherent activation mechanism for the FA pathway.\",\n      \"evidence\": \"Reconstituted deubiquitination with DNA-binding mutants of UAF1 and RAD51AP1, cellular FANCD2-Ub assays\",\n      \"pmids\": [\"31253762\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of UAF1 vs. RAD51AP1 DNA binding in vivo not quantified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Crystal and cryo-EM structures of USP1-UAF1 alone and bound to monoubiquitinated FANCI-FANCD2 revealed that UAF1 drives conformational changes in FANCI-FANCD2 to present the ubiquitinated lysine, while the USP1 N-terminus confers FANCD2 specificity; insert L1 acts as an autoinhibitory element relieved by UAF1 and DNA.\",\n      \"evidence\": \"X-ray crystallography, cryo-EM, mutagenesis of UAF1-FANCI interface, insert L1/L3 deletion mutants with biochemical activity assays, N-terminal chimera experiments\",\n      \"pmids\": [\"33795880\", \"33619839\", \"30456385\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length USP1-UAF1-DNA-PCNA complex structure not available\", \"How insert L1 communicates with the active site at atomic level not fully resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Autocleavage-defective USP1 causes replication fork stalling resolved by Spartan-mediated removal, establishing a recycling mechanism that prevents USP1 from becoming a DNA-protein crosslink-like obstacle, while cryo-EM of ML323-bound USP1 revealed a cryptic hydrophobic tunnel as the inhibitor binding site.\",\n      \"evidence\": \"Super-resolution and single-molecule tracking of autocleavage mutant; cryo-EM at 2.5 Å of USP1±ML323\",\n      \"pmids\": [\"35365626\", \"36170365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Spartan processes autocleavage-competent USP1 under normal conditions unclear\", \"Dynamics of cryptic tunnel opening not characterized\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genome-wide CRISPR screens identified RAD18 and UBE2K as required for USP1 dependency, and PCNA K164R rescued USP1 inhibitor sensitivity, definitively establishing that the cytotoxic mechanism of USP1 loss involves aberrant accumulation of ubiquitinated PCNA.\",\n      \"evidence\": \"CRISPR-Cas9 screens, PCNA WT vs K164R rescue, DNA synthesis assays\",\n      \"pmids\": [\"36228090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which downstream effectors of PCNA-Ub cause cell death not fully resolved\", \"Whether FANCD2 deubiquitination contributes to cytotoxicity in this context unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"USP1 was shown to promote PRIMPOL-generated ssDNA gap accumulation expanded by MRE11/EXO1, and RAD18-dependent PCNA-Ub was confirmed as the mechanism of USP1 inhibitor synthetic lethality in BRCA1-deficient cells, completing the mechanistic link from USP1 to replication gap-driven cell death.\",\n      \"evidence\": \"ssDNA gap assays, RAD18 KD rescue, patient-derived organoid validation of USP1 inhibitor + PARP inhibitor combination\",\n      \"pmids\": [\"38180818\", \"38885312\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ssDNA gap toxicity is the sole mechanism or synergizes with fork collapse remains open\", \"Clinical translation of USP1 inhibitor combinations awaits trial data\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"USP1 was found to suppress cGAS-STING signaling by removing K27-linked ubiquitin from SAR1A required for STING ER-to-Golgi trafficking, while separately stabilizing cGAS itself via K48-linked deubiquitination, revealing a dual and context-dependent role in innate immune regulation.\",\n      \"evidence\": \"Chemical DUB screen, K27 and K48 linkage-specific ubiquitination assays, STING trafficking, IFN reporter, in vivo viral and radiotherapy models\",\n      \"pmids\": [\"39976106\", \"38054892\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How USP1 discriminates between SAR1A and cGAS under different stimuli unknown\", \"Whether these innate immune functions require UAF1 not explicitly tested for SAR1A\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full structural understanding of how USP1-UAF1 engages PCNA on DNA, the mechanism by which USP1 selects among its growing list of substrates in different cellular contexts, and the in vivo therapeutic window of USP1 inhibitors remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of USP1-UAF1 bound to ubiquitinated PCNA on DNA\", \"Substrate prioritization mechanism across DNA repair, differentiation, and immune contexts unresolved\", \"Clinical efficacy and toxicity of USP1 inhibitors not yet established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 3, 9, 27, 28]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [17, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [10, 17]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 17, 35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 2, 5, 12, 14, 22, 27]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [4, 6, 17, 35, 36, 49, 50]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 7, 57]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16, 53, 54]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [8, 9, 27]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 15, 44]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [24, 38, 40, 45]}\n    ],\n    \"complexes\": [\n      \"USP1-UAF1\"\n    ],\n    \"partners\": [\n      \"UAF1\",\n      \"FANCD2\",\n      \"FANCI\",\n      \"PCNA\",\n      \"RAD51AP1\",\n      \"ELG1\",\n      \"ID1\",\n      \"TBK1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}