{"gene":"DHFR","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1985,"finding":"Pyrimidine dimer removal (nucleotide excision repair) occurs preferentially in the transcriptionally active DHFR gene compared to the genome overall: >67% of dimers removed from a 14.1 kb DHFR restriction fragment by 26 h post-UV irradiation vs. only ~15% genome-wide, establishing that repair efficiency is coupled to transcriptional activity of the locus.","method":"UV-endonuclease-based quantitation of pyrimidine dimers in defined restriction fragments of CHO cells amplified for DHFR","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct quantitative DNA repair assay in a well-defined genetic system, widely replicated as a foundational transcription-coupled repair finding","pmids":["3838150"],"is_preprint":false},{"year":2001,"finding":"Repression of the dhfr promoter during cell cycle withdrawal involves cooperation of two distinct complexes: (1) Sp1-pRb-HDAC1 (TSA-sensitive, histone-deacetylase-dependent) and (2) p130-E2F4-DP-1 at the E2F sites (TSA-insensitive). Co-immunoprecipitation showed HDAC1 and hypophosphorylated pRb associate with Sp1 in serum-starved cells; p130 does not co-precipitate with HDAC1. Upon phosphorylation of pRb/p130 after serum stimulation, Sp1-pRb and p130-E2F interactions are lost while Sp1-HDAC1 persists into S phase.","method":"Co-immunoprecipitation, TSA treatment, transfection reporter assays, GAL4 recruitment experiments in CHOC400 cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus multiple orthogonal reporter assays (GAL4 recruitment, TSA response) in a single focused study","pmids":["11158299"],"is_preprint":false},{"year":1996,"finding":"Genomic footprinting of the dhfr-rep3 promoter region across the cell cycle showed that Sp1 is constitutively bound to four upstream sites and does not vary during G1-to-S transition; two distinct E2F species bind the overlapping E2F sites with different sequence preferences (TTTGGCGC vs. TTTCGCGC), the latter increasing during G1-to-S transition, indicating it is the cell-cycle-regulated activator of dhfr transcription.","method":"High-resolution genomic DNase I footprinting, gel mobility shift assays with sequence-specific probes, cell cycle fractionation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genomic footprinting plus gel-shift with multiple probes, single lab, two orthogonal methods","pmids":["8552092"],"is_preprint":false},{"year":1991,"finding":"During myogenic terminal differentiation, the decrease in DHFR protein synthesis rate is fully accounted for by the decrease in DHFR mRNA levels (both drop to ~5-6% of proliferative levels with matched kinetics); DHFR mRNA remains polysomal with constant ribosome loading, indicating that translational efficiency is unchanged and post-transcriptional regulation does not contribute to DHFR downregulation in quiescent cells.","method":"Metabolic labeling of DHFR synthesis rate, Northern blot, polysome fractionation in differentiated H-αR300T myoblasts with 540 copies of amplified DHFR gene","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (synthesis rate, mRNA quantitation, polysome profiling) in a single focused study","pmids":["2046674"],"is_preprint":false},{"year":2009,"finding":"DHFR mediates folic acid (FA)-induced improvement of endothelial NO and tetrahydrobiopterin (H4B) bioavailability: FA upregulates DHFR expression and activity; DHFR converts dihydrofolic acid to tetrahydrofolate (THF) as demonstrated by HPLC fluorescent assay; siRNA knockdown of DHFR or methotrexate pre-treatment abolishes FA-induced restoration of NO levels in angiotensin II-treated cells, establishing DHFR as a required intermediate in FA-dependent vascular protection.","method":"HPLC-fluorescence DHFR activity assay, DHFR siRNA knockdown, methotrexate inhibition, superoxide and NO measurements in endothelial cells and Ang II-infused mice","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (enzymatic assay, siRNA, pharmacological inhibition, in vivo model) in a single focused study","pmids":["19660467"],"is_preprint":false},{"year":2011,"finding":"Homozygous DHFR mutation p.Asp153Val causes severely reduced DHFR enzymatic activity and reduced DHFR protein expression (not mRNA), leading to megaloblastic anemia and cerebral folate deficiency with neurologic disease; heterozygous cells show intermediate DHFR activity, confirming a gene-dosage effect. DHFR is required to maintain sufficient CSF and RBC folate levels even when plasma folate is normal.","method":"DHFR enzyme activity assay and fluorescein-labeled methotrexate (FMTX) binding in EBV-immortalized lymphoblastoid cells, RT-PCR, protein expression analysis, genome-wide homozygosity mapping, liquid chromatography-tandem MS of folate profile","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct enzyme activity assay with mutant protein plus multiple orthogonal methods (FMTX binding, protein vs. mRNA analysis, patient metabolomics) and clinical confirmation","pmids":["21310277"],"is_preprint":false},{"year":2020,"finding":"In Marfan syndrome mice, TGFβ signaling downregulates DHFR protein, uncoupling eNOS and promoting aortic aneurysm formation via a TGFβ/NOX4/DHFR/eNOS-uncoupling feed-forward loop. Anti-TGFβ treatment restored DHFR abundance and recoupled eNOS; folic acid upregulated endothelial DHFR expression and activity to restore H4B, recouple eNOS, and attenuate aneurysm formation. A NO scavenger (PTIO) reversed FA effects on the TGFβ/NOX4 axis, placing DHFR-mediated NO production upstream of TGFβ suppression.","method":"In vivo Fbn1C1039G/+ mouse model, anti-TGFβ antibody treatment, folic acid diet, RNAi in human aortic endothelial cells, DHFR activity assay, H4B/ROS measurements, aortic root measurements","journal":"Redox biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vivo genetics, RNAi, pharmacological rescue, enzymatic assay, NO scavenger epistasis) in a single focused study","pmids":["33126053"],"is_preprint":false},{"year":1999,"finding":"RNA synthesis from the DHFR gene recovers significantly faster after UV irradiation than can be accounted for by removal of photolesions from the transcribed strand, demonstrating that RNA polymerase II can bypass UV-induced lesions in the DHFR gene prior to their repair.","method":"In vivo RNA labeling and nuclear run-on transcription assays at three positions within the DHFR gene in UV-irradiated CHO cells, compared to published photolesion removal kinetics","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — two orthogonal transcription assays, single lab, comparison with published data from a different study","pmids":["10190552"],"is_preprint":false},{"year":2015,"finding":"Hydride transfer by E. coli DHFR (ecDHFR; ortholog informative for mechanism) is independent of protein mass: heavy ecDHFR (uniformly labeled with 13C/15N/2H) does not show altered hydride transfer rate constants in refined kinetic and computational (transition path sampling) experiments, but does show faster substrate dissociation. Fast femtosecond-to-picosecond protein motions are not coupled to the chemical (hydride transfer) step.","method":"Transition path sampling simulations, kinetic isotope effect measurements, heavy enzyme studies (13C/15N/2H labeling), pre-steady-state kinetics","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — integrated in vitro enzyme kinetics with isotope labeling and transition path sampling computation, orthogonal methods in a single rigorous study","pmids":["26652185"],"is_preprint":false},{"year":2018,"finding":"Asp122 in E. coli DHFR (conserved across the DHFR family) is required for optimal hydride transfer: Asp122X mutations (Asn, Ser, Ala) reduce the hydride transfer rate by perturbing coupled protein motions along the reaction coordinate. D122N and D122S inhibit coupled motion while D122A enhances it, yet all three mutations similarly lower catalytic rate, demonstrating a Goldilocks principle of enzyme flexibility for DHFR catalysis.","method":"Computational mutagenesis (QM/MM), molecular dynamics simulations, correlated/principal component motion analysis, enzyme kinetics","journal":"The journal of physical chemistry. B","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous computational study with mutagenesis simulation, but primarily in silico without in vitro validation of the mutants in this paper","pmids":["30040418"],"is_preprint":false},{"year":2007,"finding":"Ligand binding to DHFR produces 'network-bridging effects': systematic residue interaction network analysis and molecular dynamics of native and 19 circularly permuted DHFRs show that ligand binding at the active site causes most interaction network paths to pass through the cofactor, shortening average shortest path lengths. The active site coincides with residues of highest network centrality, and chain cleavage in folding element regions near the active site deactivates DHFR by large perturbations in network properties.","method":"Residue interaction network analysis, molecular dynamics simulations of native DHFR and 19 circularly permuted variants, network centrality calculations","journal":"PLoS computational biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — computational study with systematic variant analysis, single approach, no in vitro experimental validation","pmids":["17571919"],"is_preprint":false},{"year":2023,"finding":"MTX-based PROTACs achieve proteasome- and E3 ligase-dependent selective degradation of DHFR in multiple cancer cell lines. Unlike MTX treatment (which increases cellular DHFR protein expression), DHFR-targeting PROTACs produce distinct, less-lethal phenotypes, demonstrating that DHFR degradation has different functional consequences from DHFR inhibition.","method":"PROTAC synthesis, cell viability assays, Western blot for DHFR degradation, proteasome inhibitor controls, E3 ligase-dependent activity confirmation in cancer cell lines","journal":"Cell chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based PROTAC degradation with mechanistic controls (proteasome inhibition, E3 ligase dependence), single lab, multiple orthogonal readouts","pmids":["37875111"],"is_preprint":false},{"year":2021,"finding":"DHFR inhibition (by methotrexate or EphB activation with synthetic ligands) reduces the self-renewal capacity and tumorigenic potential of human brain tumor initiating cells (BTIC) both in vitro (sphere formation) and in a cerebral organoid glioma model, establishing DHFR activity as required for BTIC self-renewal in one-carbon metabolism.","method":"MTX treatment, EphB synthetic ligand activation, sphere formation assay, cerebral organoid glioma (GLICO) model, four human BTIC lines","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent DHFR inhibition approaches (MTX and EphB ligand), multiple BTIC lines and in vivo organoid model, single lab","pmids":["33545223"],"is_preprint":false},{"year":1993,"finding":"Removal of MMS-induced N-methylpurines (base excision repair) is not preferential in the transcriptionally active DHFR gene compared to a non-transcribed flanking region or mitochondrial DNA; repair rates are similar across all three regions, demonstrating that transcription-coupled repair does not apply to N-methylpurine adducts in the DHFR locus.","method":"Alkaline hydrolysis generating strand breaks at AP sites after neutral depurination, quantitation in DHFR gene domain, 3'-flanking region, and mitochondrial DNA in CHO-B11 cells","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct biochemical assay in defined loci, negative result (no preferential repair), single lab","pmids":["8222061"],"is_preprint":false},{"year":2000,"finding":"RIP60, a 15-zinc-finger protein that binds ATT-rich sites in the dhfr replication origin oribeta, forms homodimers that loop a 720 bp DNA region in vitro. Structural dissection showed that zinc finger hands Z1 and Z2 independently bind oribeta sites with different affinities; hand Z2 plus part of a proline-rich region is sufficient for protein multimerization and DNA looping. RIP60 has weak replication enhancer activity in plasmid replication assays.","method":"One-hybrid screen for human RIP60 cDNA, gel mobility shift assays, DNase I footprinting, ligation enhancement assay, atomic force microscopy, polyomavirus origin-dependent replication assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods (footprinting, gel shift, AFM, replication assay) characterizing RIP60 interaction at the DHFR origin, single lab","pmids":["10606657"],"is_preprint":false}],"current_model":"DHFR is a metabolic enzyme that catalyzes NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, a reaction essential for one-carbon metabolism, purine/pyrimidine biosynthesis, and maintenance of tetrahydrobiopterin (H4B) levels; its transcription is cell-cycle regulated through cooperative Sp1-pRb-HDAC1 and p130-E2F4 complexes at its promoter, its hydride transfer mechanism involves active-site network communication and appropriately tuned protein flexibility (Goldilocks principle), and in endothelial cells DHFR activity is required for folic acid-mediated eNOS recoupling and vascular NO bioavailability via H4B regeneration, while in stem-like cells DHFR supports self-renewal through one-carbon metabolism."},"narrative":{"mechanistic_narrative":"DHFR is an NADPH-dependent oxidoreductase central to one-carbon metabolism, catalyzing reduction of dihydrofolate to tetrahydrofolate to maintain cellular folate pools required for purine/pyrimidine synthesis and downstream physiology [PMID:21310277]. Loss of function has direct clinical consequences: a homozygous p.Asp153Val mutation severely reduces DHFR enzymatic activity and protein (but not mRNA) levels, causing megaloblastic anemia and cerebral folate deficiency, and demonstrating that DHFR is needed to maintain CSF and red-cell folate even when plasma folate is adequate [PMID:21310277]. In the vascular endothelium DHFR regenerates tetrahydrobiopterin (H4B) from folic acid-derived substrate, and this activity is required for eNOS recoupling and nitric oxide bioavailability; DHFR knockdown or methotrexate abolishes folic-acid-mediated restoration of NO, and TGFβ-driven DHFR downregulation uncouples eNOS to promote aortic aneurysm in a feed-forward loop reversible by folic acid [PMID:19660467, PMID:33126053]. DHFR activity also supports self-renewal and tumorigenicity of brain tumor-initiating cells through one-carbon metabolism [PMID:33545223]. At the catalytic level, mechanistic studies in the bacterial ortholog establish that hydride transfer is independent of overall protein mass but depends on appropriately tuned, residue-specific coupled motions—a conserved active-site residue (Asp122) is required for optimal catalysis, illustrating a Goldilocks principle of enzyme flexibility, and ligand binding reorganizes the residue interaction network around the active site [PMID:26652185, PMID:30040418, PMID:17571919]. DHFR transcription is cell-cycle regulated: Sp1 is constitutively bound to upstream promoter sites while a cell-cycle-regulated E2F species activates transcription at G1-to-S, and promoter repression upon quiescence is enforced by cooperative Sp1-pRb-HDAC1 and p130-E2F4-DP1 complexes [PMID:11158299, PMID:8552092]. The amplified DHFR locus has also served as a model for transcription-coupled DNA repair, where pyrimidine dimer removal is preferential in the active gene [PMID:3838150]. Pharmacologically, DHFR can be eliminated by methotrexate-based PROTACs, whose degradation phenotype differs from enzymatic inhibition [PMID:37875111].","teleology":[{"year":1985,"claim":"Established that DNA repair efficiency is coupled to transcriptional activity using the amplified DHFR locus as a tractable model, answering whether active genes are repaired preferentially.","evidence":"UV-endonuclease quantitation of pyrimidine dimers in defined DHFR restriction fragments in amplified CHO cells","pmids":["3838150"],"confidence":"High","gaps":["Does not identify the repair factors mediating preferential repair","Specific to UV pyrimidine dimers, not generalizable to all lesion types"]},{"year":1991,"claim":"Resolved whether DHFR downregulation in quiescent/differentiated cells is transcriptional or post-transcriptional, showing it is fully accounted for by reduced mRNA with unchanged translational efficiency.","evidence":"Metabolic labeling, Northern blot, and polysome fractionation in differentiated myoblasts with amplified DHFR","pmids":["2046674"],"confidence":"High","gaps":["Does not define the transcription factors driving the mRNA decrease","Limited to myogenic differentiation context"]},{"year":1993,"claim":"Tested whether transcription-coupled repair is lesion-general, showing N-methylpurine base-excision repair is NOT preferential in the active DHFR gene, delimiting the scope of transcription-coupled repair.","evidence":"Alkaline hydrolysis quantitation of AP sites at DHFR, flanking, and mitochondrial DNA in CHO cells","pmids":["8222061"],"confidence":"Medium","gaps":["Negative result; single lab","Does not address other base lesions or repair pathways"]},{"year":1996,"claim":"Defined the cell-cycle architecture of the DHFR promoter, distinguishing constitutive Sp1 binding from a cell-cycle-regulated E2F species that activates transcription at G1-to-S.","evidence":"High-resolution genomic DNase I footprinting and gel mobility shift with sequence-specific probes across the cell cycle","pmids":["8552092"],"confidence":"High","gaps":["Does not identify the specific E2F family member by isoform","Footprinting in CHO amplified locus may not capture single-copy regulation"]},{"year":1999,"claim":"Asked whether transcription resumes only after lesion repair, showing RNA polymerase II can bypass UV lesions in DHFR before they are removed.","evidence":"In vivo RNA labeling and nuclear run-on at three DHFR positions in UV-irradiated CHO cells","pmids":["10190552"],"confidence":"Medium","gaps":["Compared to published photolesion kinetics from a separate study rather than matched controls","Mechanism of polymerase bypass not defined"]},{"year":2000,"claim":"Characterized RIP60 as a zinc-finger protein binding the DHFR replication origin oribeta, looping DNA via defined zinc-finger hands, linking the locus to origin function.","evidence":"One-hybrid cloning, footprinting, gel shift, AFM, and origin-dependent replication assay","pmids":["10606657"],"confidence":"Medium","gaps":["RIP60 replication enhancer activity is weak; physiological role at oribeta unproven","Does not establish requirement for DHFR origin firing in vivo"]},{"year":2001,"claim":"Mechanistically dissected DHFR promoter repression during cell-cycle withdrawal into two cooperating complexes (Sp1-pRb-HDAC1 and p130-E2F4-DP1), explaining how histone deacetylation and pocket proteins coordinate repression.","evidence":"Reciprocal Co-IP, TSA treatment, reporter assays, and GAL4 recruitment in CHOC400 cells","pmids":["11158299"],"confidence":"High","gaps":["Does not map chromatin changes at the endogenous single-copy promoter genome-wide","Quantitative contribution of each complex to repression unresolved"]},{"year":2007,"claim":"Addressed how the active site coordinates catalysis at the level of protein structure, showing ligand binding reorganizes the residue interaction network through the cofactor and that active-site residues have highest network centrality.","evidence":"Residue interaction network analysis and molecular dynamics of native and 19 circularly permuted DHFRs","pmids":["17571919"],"confidence":"Medium","gaps":["Computational only; no in vitro validation of permuted variants","Network predictions not tied to measured catalytic parameters"]},{"year":2009,"claim":"Established DHFR as the required intermediate for folic-acid-mediated endothelial protection, regenerating H4B to recouple eNOS and restore NO.","evidence":"HPLC-fluorescence DHFR activity assay, siRNA knockdown, methotrexate inhibition, and NO/superoxide measurements in endothelial cells and Ang II mice","pmids":["19660467"],"confidence":"High","gaps":["Does not define how folic acid upregulates DHFR expression","Tissue specificity of the H4B-regeneration role not delineated"]},{"year":2015,"claim":"Tested whether fast protein motions drive the chemical step, showing hydride transfer in the bacterial ortholog is mass-independent and uncoupled from femtosecond-picosecond motions.","evidence":"Heavy-enzyme (13C/15N/2H) kinetics, kinetic isotope effects, and transition path sampling in ecDHFR","pmids":["26652185"],"confidence":"High","gaps":["Performed in E. coli ortholog; human enzyme not tested","Does not exclude slower conformational coupling to product release"]},{"year":2018,"claim":"Identified a conserved residue (Asp122) tuning catalytically productive flexibility, establishing a Goldilocks principle whereby both excess and deficient coupled motion impair hydride transfer.","evidence":"QM/MM, molecular dynamics, correlated-motion analysis, and kinetics of Asp122 mutants in ecDHFR","pmids":["30040418"],"confidence":"Medium","gaps":["Primarily in silico; mutant kinetics not validated in vitro in this study","Human DHFR equivalent residue not directly tested"]},{"year":2011,"claim":"Connected DHFR loss-of-function to human disease, showing a homozygous mutation reduces enzyme activity/protein and causes megaloblastic anemia and cerebral folate deficiency with gene-dosage effect.","evidence":"DHFR enzyme activity and FMTX-binding assays in patient lymphoblastoid cells, homozygosity mapping, and LC-MS/MS folate profiling","pmids":["21310277"],"confidence":"High","gaps":["Single mutation/family; allelic spectrum not defined","Mechanism of selective protein destabilization not resolved"]},{"year":2021,"claim":"Demonstrated a role for DHFR-dependent one-carbon metabolism in cancer stem-like cell biology, showing inhibition reduces brain tumor-initiating cell self-renewal and tumorigenicity.","evidence":"Methotrexate and EphB ligand inhibition, sphere formation, and cerebral organoid glioma model with four BTIC lines","pmids":["33545223"],"confidence":"Medium","gaps":["Two inhibition approaches but no genetic DHFR knockout validation","Downstream one-carbon metabolites mediating self-renewal not identified"]},{"year":2023,"claim":"Distinguished DHFR degradation from inhibition, showing MTX-based PROTACs degrade DHFR via proteasome/E3 ligase and yield distinct phenotypes from enzymatic inhibition.","evidence":"PROTAC synthesis, Western blot degradation, proteasome and E3-ligase dependence controls in cancer cell lines","pmids":["37875111"],"confidence":"Medium","gaps":["Why degradation is less lethal than inhibition is unexplained","In vivo efficacy and selectivity not established"]},{"year":null,"claim":"How DHFR catalytic, transcriptional, and metabolic regulation integrate in human tissues—and whether the bacterial-ortholog flexibility/Goldilocks mechanisms hold for human DHFR—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Hydride-transfer flexibility findings derive from E. coli ortholog, not validated in human DHFR","No structural model of human DHFR regulatory complexes at the promoter","Mechanism linking folic acid sensing to DHFR transcriptional/protein upregulation undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[4,5,6]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[8,9]}],"localization":[],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[4,5,12]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,2]}],"complexes":[],"partners":[],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P00374","full_name":"Dihydrofolate reductase","aliases":[],"length_aa":187,"mass_kda":21.5,"function":"Catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate in a NADPH-dependent manner (PubMed:12096917, PubMed:15039552, PubMed:17569517, PubMed:19196009, PubMed:19478082, PubMed:21876184, PubMed:9719595). Key enzyme in folate metabolism. Contributes to the nuclear and mitochondrial de novo thymidylate biosynthesis pathway (PubMed:21876188, PubMed:22235121). Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Binds its own mRNA and that of DHFR2","subcellular_location":"Mitochondrion; Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/P00374/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DHFR","classification":"Common Essential","n_dependent_lines":779,"n_total_lines":1090,"dependency_fraction":0.7146788990825688},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ELOVL1","stoichiometry":0.2},{"gene":"SAR1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DHFR","total_profiled":1310},"omim":[{"mim_id":"619498","title":"ZINC FINGER CCCH DOMAIN-CONTAINING PROTEIN 4; ZC3H4","url":"https://www.omim.org/entry/619498"},{"mim_id":"619039","title":"REPLICATION INITIATOR 1; REPIN1","url":"https://www.omim.org/entry/619039"},{"mim_id":"616588","title":"DIHYDROFOLATE REDUCTASE-LIKE 1; DHFRL1","url":"https://www.omim.org/entry/616588"},{"mim_id":"616368","title":"CHOPS SYNDROME; CHOPS","url":"https://www.omim.org/entry/616368"},{"mim_id":"613839","title":"MEGALOBLASTIC ANEMIA DUE TO DIHYDROFOLATE REDUCTASE DEFICIENCY","url":"https://www.omim.org/entry/613839"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DHFR"},"hgnc":{"alias_symbol":["DHFR1"],"prev_symbol":[]},"alphafold":{"accession":"P00374","domains":[{"cath_id":"3.40.430.10","chopping":"4-177","consensus_level":"medium","plddt":96.9252,"start":4,"end":177}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P00374","model_url":"https://alphafold.ebi.ac.uk/files/AF-P00374-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P00374-F1-predicted_aligned_error_v6.png","plddt_mean":96.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DHFR","jax_strain_url":"https://www.jax.org/strain/search?query=DHFR"},"sequence":{"accession":"P00374","fasta_url":"https://rest.uniprot.org/uniprotkb/P00374.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P00374/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P00374"}},"corpus_meta":[{"pmid":"3838150","id":"PMC_3838150","title":"DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall.","date":"1985","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/3838150","citation_count":1141,"is_preprint":false},{"pmid":"30909399","id":"PMC_30909399","title":"DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents.","date":"2019","source":"Molecules (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/30909399","citation_count":157,"is_preprint":false},{"pmid":"2674679","id":"PMC_2674679","title":"Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene.","date":"1989","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/2674679","citation_count":120,"is_preprint":false},{"pmid":"15718161","id":"PMC_15718161","title":"Targeting DHFR in parasitic protozoa.","date":"2005","source":"Drug discovery today","url":"https://pubmed.ncbi.nlm.nih.gov/15718161","citation_count":105,"is_preprint":false},{"pmid":"17283199","id":"PMC_17283199","title":"Common origin and fixation of Plasmodium falciparum dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance in a low-transmission area in South America.","date":"2007","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/17283199","citation_count":102,"is_preprint":false},{"pmid":"3018531","id":"PMC_3018531","title":"Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.","date":"1985","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/3018531","citation_count":99,"is_preprint":false},{"pmid":"19660467","id":"PMC_19660467","title":"Mechanistic insights into folic acid-dependent vascular protection: dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: a novel HPLC-based fluorescent assay for DHFR activity.","date":"2009","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/19660467","citation_count":92,"is_preprint":false},{"pmid":"21310277","id":"PMC_21310277","title":"Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease.","date":"2011","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/21310277","citation_count":81,"is_preprint":false},{"pmid":"18471065","id":"PMC_18471065","title":"Emergence of a dhfr mutation conferring high-level drug resistance in Plasmodium falciparum populations from southwest Uganda.","date":"2008","source":"The Journal of infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/18471065","citation_count":79,"is_preprint":false},{"pmid":"8570205","id":"PMC_8570205","title":"c-Myc overexpression associated DHFR gene 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MCP","url":"https://pubmed.ncbi.nlm.nih.gov/29203496","citation_count":10,"is_preprint":false},{"pmid":"31031194","id":"PMC_31031194","title":"Establishment of DHFR-deficient HEK293 cells for high yield of therapeutic glycoproteins.","date":"2019","source":"Journal of bioscience and bioengineering","url":"https://pubmed.ncbi.nlm.nih.gov/31031194","citation_count":9,"is_preprint":false},{"pmid":"16164748","id":"PMC_16164748","title":"Concurrence of Plasmodium falciparum dhfr and crt mutations in northern Ghana.","date":"2005","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/16164748","citation_count":9,"is_preprint":false},{"pmid":"36469541","id":"PMC_36469541","title":"Disparate selection of mutations in the dihydrofolate reductase gene (dhfr) of Plasmodium ovale curtisi and P. o. wallikeri in Africa.","date":"2022","source":"PLoS neglected tropical diseases","url":"https://pubmed.ncbi.nlm.nih.gov/36469541","citation_count":9,"is_preprint":false},{"pmid":"37243214","id":"PMC_37243214","title":"DHFR Inhibitors Display a Pleiotropic Anti-Viral Activity against SARS-CoV-2: Insights into the Mechanisms of Action.","date":"2023","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/37243214","citation_count":9,"is_preprint":false},{"pmid":"8088538","id":"PMC_8088538","title":"The isolation and characterization of the gene (dfr1) encoding dihydrofolate reductase (DHFR) in Schizosaccharomyces pombe.","date":"1994","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/8088538","citation_count":9,"is_preprint":false},{"pmid":"26221921","id":"PMC_26221921","title":"The 19-bp deletion polymorphism of dihydrofolate reductase (DHFR) and nonsyndromic cleft lip with or without cleft palate: evidence for a protective role.","date":"2015","source":"Journal of applied oral science : revista FOB","url":"https://pubmed.ncbi.nlm.nih.gov/26221921","citation_count":9,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53016,"output_tokens":4312,"usd":0.111864,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12314,"output_tokens":4420,"usd":0.086035,"stage2_stop_reason":"end_turn"},"total_usd":0.197899,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1985,\n      \"finding\": \"Pyrimidine dimer removal (nucleotide excision repair) occurs preferentially in the transcriptionally active DHFR gene compared to the genome overall: >67% of dimers removed from a 14.1 kb DHFR restriction fragment by 26 h post-UV irradiation vs. only ~15% genome-wide, establishing that repair efficiency is coupled to transcriptional activity of the locus.\",\n      \"method\": \"UV-endonuclease-based quantitation of pyrimidine dimers in defined restriction fragments of CHO cells amplified for DHFR\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct quantitative DNA repair assay in a well-defined genetic system, widely replicated as a foundational transcription-coupled repair finding\",\n      \"pmids\": [\"3838150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Repression of the dhfr promoter during cell cycle withdrawal involves cooperation of two distinct complexes: (1) Sp1-pRb-HDAC1 (TSA-sensitive, histone-deacetylase-dependent) and (2) p130-E2F4-DP-1 at the E2F sites (TSA-insensitive). Co-immunoprecipitation showed HDAC1 and hypophosphorylated pRb associate with Sp1 in serum-starved cells; p130 does not co-precipitate with HDAC1. Upon phosphorylation of pRb/p130 after serum stimulation, Sp1-pRb and p130-E2F interactions are lost while Sp1-HDAC1 persists into S phase.\",\n      \"method\": \"Co-immunoprecipitation, TSA treatment, transfection reporter assays, GAL4 recruitment experiments in CHOC400 cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus multiple orthogonal reporter assays (GAL4 recruitment, TSA response) in a single focused study\",\n      \"pmids\": [\"11158299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Genomic footprinting of the dhfr-rep3 promoter region across the cell cycle showed that Sp1 is constitutively bound to four upstream sites and does not vary during G1-to-S transition; two distinct E2F species bind the overlapping E2F sites with different sequence preferences (TTTGGCGC vs. TTTCGCGC), the latter increasing during G1-to-S transition, indicating it is the cell-cycle-regulated activator of dhfr transcription.\",\n      \"method\": \"High-resolution genomic DNase I footprinting, gel mobility shift assays with sequence-specific probes, cell cycle fractionation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genomic footprinting plus gel-shift with multiple probes, single lab, two orthogonal methods\",\n      \"pmids\": [\"8552092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"During myogenic terminal differentiation, the decrease in DHFR protein synthesis rate is fully accounted for by the decrease in DHFR mRNA levels (both drop to ~5-6% of proliferative levels with matched kinetics); DHFR mRNA remains polysomal with constant ribosome loading, indicating that translational efficiency is unchanged and post-transcriptional regulation does not contribute to DHFR downregulation in quiescent cells.\",\n      \"method\": \"Metabolic labeling of DHFR synthesis rate, Northern blot, polysome fractionation in differentiated H-αR300T myoblasts with 540 copies of amplified DHFR gene\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (synthesis rate, mRNA quantitation, polysome profiling) in a single focused study\",\n      \"pmids\": [\"2046674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DHFR mediates folic acid (FA)-induced improvement of endothelial NO and tetrahydrobiopterin (H4B) bioavailability: FA upregulates DHFR expression and activity; DHFR converts dihydrofolic acid to tetrahydrofolate (THF) as demonstrated by HPLC fluorescent assay; siRNA knockdown of DHFR or methotrexate pre-treatment abolishes FA-induced restoration of NO levels in angiotensin II-treated cells, establishing DHFR as a required intermediate in FA-dependent vascular protection.\",\n      \"method\": \"HPLC-fluorescence DHFR activity assay, DHFR siRNA knockdown, methotrexate inhibition, superoxide and NO measurements in endothelial cells and Ang II-infused mice\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (enzymatic assay, siRNA, pharmacological inhibition, in vivo model) in a single focused study\",\n      \"pmids\": [\"19660467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Homozygous DHFR mutation p.Asp153Val causes severely reduced DHFR enzymatic activity and reduced DHFR protein expression (not mRNA), leading to megaloblastic anemia and cerebral folate deficiency with neurologic disease; heterozygous cells show intermediate DHFR activity, confirming a gene-dosage effect. DHFR is required to maintain sufficient CSF and RBC folate levels even when plasma folate is normal.\",\n      \"method\": \"DHFR enzyme activity assay and fluorescein-labeled methotrexate (FMTX) binding in EBV-immortalized lymphoblastoid cells, RT-PCR, protein expression analysis, genome-wide homozygosity mapping, liquid chromatography-tandem MS of folate profile\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct enzyme activity assay with mutant protein plus multiple orthogonal methods (FMTX binding, protein vs. mRNA analysis, patient metabolomics) and clinical confirmation\",\n      \"pmids\": [\"21310277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In Marfan syndrome mice, TGFβ signaling downregulates DHFR protein, uncoupling eNOS and promoting aortic aneurysm formation via a TGFβ/NOX4/DHFR/eNOS-uncoupling feed-forward loop. Anti-TGFβ treatment restored DHFR abundance and recoupled eNOS; folic acid upregulated endothelial DHFR expression and activity to restore H4B, recouple eNOS, and attenuate aneurysm formation. A NO scavenger (PTIO) reversed FA effects on the TGFβ/NOX4 axis, placing DHFR-mediated NO production upstream of TGFβ suppression.\",\n      \"method\": \"In vivo Fbn1C1039G/+ mouse model, anti-TGFβ antibody treatment, folic acid diet, RNAi in human aortic endothelial cells, DHFR activity assay, H4B/ROS measurements, aortic root measurements\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vivo genetics, RNAi, pharmacological rescue, enzymatic assay, NO scavenger epistasis) in a single focused study\",\n      \"pmids\": [\"33126053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"RNA synthesis from the DHFR gene recovers significantly faster after UV irradiation than can be accounted for by removal of photolesions from the transcribed strand, demonstrating that RNA polymerase II can bypass UV-induced lesions in the DHFR gene prior to their repair.\",\n      \"method\": \"In vivo RNA labeling and nuclear run-on transcription assays at three positions within the DHFR gene in UV-irradiated CHO cells, compared to published photolesion removal kinetics\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — two orthogonal transcription assays, single lab, comparison with published data from a different study\",\n      \"pmids\": [\"10190552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Hydride transfer by E. coli DHFR (ecDHFR; ortholog informative for mechanism) is independent of protein mass: heavy ecDHFR (uniformly labeled with 13C/15N/2H) does not show altered hydride transfer rate constants in refined kinetic and computational (transition path sampling) experiments, but does show faster substrate dissociation. Fast femtosecond-to-picosecond protein motions are not coupled to the chemical (hydride transfer) step.\",\n      \"method\": \"Transition path sampling simulations, kinetic isotope effect measurements, heavy enzyme studies (13C/15N/2H labeling), pre-steady-state kinetics\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — integrated in vitro enzyme kinetics with isotope labeling and transition path sampling computation, orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"26652185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Asp122 in E. coli DHFR (conserved across the DHFR family) is required for optimal hydride transfer: Asp122X mutations (Asn, Ser, Ala) reduce the hydride transfer rate by perturbing coupled protein motions along the reaction coordinate. D122N and D122S inhibit coupled motion while D122A enhances it, yet all three mutations similarly lower catalytic rate, demonstrating a Goldilocks principle of enzyme flexibility for DHFR catalysis.\",\n      \"method\": \"Computational mutagenesis (QM/MM), molecular dynamics simulations, correlated/principal component motion analysis, enzyme kinetics\",\n      \"journal\": \"The journal of physical chemistry. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous computational study with mutagenesis simulation, but primarily in silico without in vitro validation of the mutants in this paper\",\n      \"pmids\": [\"30040418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Ligand binding to DHFR produces 'network-bridging effects': systematic residue interaction network analysis and molecular dynamics of native and 19 circularly permuted DHFRs show that ligand binding at the active site causes most interaction network paths to pass through the cofactor, shortening average shortest path lengths. The active site coincides with residues of highest network centrality, and chain cleavage in folding element regions near the active site deactivates DHFR by large perturbations in network properties.\",\n      \"method\": \"Residue interaction network analysis, molecular dynamics simulations of native DHFR and 19 circularly permuted variants, network centrality calculations\",\n      \"journal\": \"PLoS computational biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — computational study with systematic variant analysis, single approach, no in vitro experimental validation\",\n      \"pmids\": [\"17571919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MTX-based PROTACs achieve proteasome- and E3 ligase-dependent selective degradation of DHFR in multiple cancer cell lines. Unlike MTX treatment (which increases cellular DHFR protein expression), DHFR-targeting PROTACs produce distinct, less-lethal phenotypes, demonstrating that DHFR degradation has different functional consequences from DHFR inhibition.\",\n      \"method\": \"PROTAC synthesis, cell viability assays, Western blot for DHFR degradation, proteasome inhibitor controls, E3 ligase-dependent activity confirmation in cancer cell lines\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based PROTAC degradation with mechanistic controls (proteasome inhibition, E3 ligase dependence), single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"37875111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DHFR inhibition (by methotrexate or EphB activation with synthetic ligands) reduces the self-renewal capacity and tumorigenic potential of human brain tumor initiating cells (BTIC) both in vitro (sphere formation) and in a cerebral organoid glioma model, establishing DHFR activity as required for BTIC self-renewal in one-carbon metabolism.\",\n      \"method\": \"MTX treatment, EphB synthetic ligand activation, sphere formation assay, cerebral organoid glioma (GLICO) model, four human BTIC lines\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent DHFR inhibition approaches (MTX and EphB ligand), multiple BTIC lines and in vivo organoid model, single lab\",\n      \"pmids\": [\"33545223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Removal of MMS-induced N-methylpurines (base excision repair) is not preferential in the transcriptionally active DHFR gene compared to a non-transcribed flanking region or mitochondrial DNA; repair rates are similar across all three regions, demonstrating that transcription-coupled repair does not apply to N-methylpurine adducts in the DHFR locus.\",\n      \"method\": \"Alkaline hydrolysis generating strand breaks at AP sites after neutral depurination, quantitation in DHFR gene domain, 3'-flanking region, and mitochondrial DNA in CHO-B11 cells\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct biochemical assay in defined loci, negative result (no preferential repair), single lab\",\n      \"pmids\": [\"8222061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"RIP60, a 15-zinc-finger protein that binds ATT-rich sites in the dhfr replication origin oribeta, forms homodimers that loop a 720 bp DNA region in vitro. Structural dissection showed that zinc finger hands Z1 and Z2 independently bind oribeta sites with different affinities; hand Z2 plus part of a proline-rich region is sufficient for protein multimerization and DNA looping. RIP60 has weak replication enhancer activity in plasmid replication assays.\",\n      \"method\": \"One-hybrid screen for human RIP60 cDNA, gel mobility shift assays, DNase I footprinting, ligation enhancement assay, atomic force microscopy, polyomavirus origin-dependent replication assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods (footprinting, gel shift, AFM, replication assay) characterizing RIP60 interaction at the DHFR origin, single lab\",\n      \"pmids\": [\"10606657\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DHFR is a metabolic enzyme that catalyzes NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, a reaction essential for one-carbon metabolism, purine/pyrimidine biosynthesis, and maintenance of tetrahydrobiopterin (H4B) levels; its transcription is cell-cycle regulated through cooperative Sp1-pRb-HDAC1 and p130-E2F4 complexes at its promoter, its hydride transfer mechanism involves active-site network communication and appropriately tuned protein flexibility (Goldilocks principle), and in endothelial cells DHFR activity is required for folic acid-mediated eNOS recoupling and vascular NO bioavailability via H4B regeneration, while in stem-like cells DHFR supports self-renewal through one-carbon metabolism.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DHFR is an NADPH-dependent oxidoreductase central to one-carbon metabolism, catalyzing reduction of dihydrofolate to tetrahydrofolate to maintain cellular folate pools required for purine/pyrimidine synthesis and downstream physiology [#5]. Loss of function has direct clinical consequences: a homozygous p.Asp153Val mutation severely reduces DHFR enzymatic activity and protein (but not mRNA) levels, causing megaloblastic anemia and cerebral folate deficiency, and demonstrating that DHFR is needed to maintain CSF and red-cell folate even when plasma folate is adequate [#5]. In the vascular endothelium DHFR regenerates tetrahydrobiopterin (H4B) from folic acid-derived substrate, and this activity is required for eNOS recoupling and nitric oxide bioavailability; DHFR knockdown or methotrexate abolishes folic-acid-mediated restoration of NO, and TGFβ-driven DHFR downregulation uncouples eNOS to promote aortic aneurysm in a feed-forward loop reversible by folic acid [#4, #6]. DHFR activity also supports self-renewal and tumorigenicity of brain tumor-initiating cells through one-carbon metabolism [#12]. At the catalytic level, mechanistic studies in the bacterial ortholog establish that hydride transfer is independent of overall protein mass but depends on appropriately tuned, residue-specific coupled motions—a conserved active-site residue (Asp122) is required for optimal catalysis, illustrating a Goldilocks principle of enzyme flexibility, and ligand binding reorganizes the residue interaction network around the active site [#8, #9, #10]. DHFR transcription is cell-cycle regulated: Sp1 is constitutively bound to upstream promoter sites while a cell-cycle-regulated E2F species activates transcription at G1-to-S, and promoter repression upon quiescence is enforced by cooperative Sp1-pRb-HDAC1 and p130-E2F4-DP1 complexes [#1, #2]. The amplified DHFR locus has also served as a model for transcription-coupled DNA repair, where pyrimidine dimer removal is preferential in the active gene [#0]. Pharmacologically, DHFR can be eliminated by methotrexate-based PROTACs, whose degradation phenotype differs from enzymatic inhibition [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Established that DNA repair efficiency is coupled to transcriptional activity using the amplified DHFR locus as a tractable model, answering whether active genes are repaired preferentially.\",\n      \"evidence\": \"UV-endonuclease quantitation of pyrimidine dimers in defined DHFR restriction fragments in amplified CHO cells\",\n      \"pmids\": [\"3838150\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the repair factors mediating preferential repair\", \"Specific to UV pyrimidine dimers, not generalizable to all lesion types\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Resolved whether DHFR downregulation in quiescent/differentiated cells is transcriptional or post-transcriptional, showing it is fully accounted for by reduced mRNA with unchanged translational efficiency.\",\n      \"evidence\": \"Metabolic labeling, Northern blot, and polysome fractionation in differentiated myoblasts with amplified DHFR\",\n      \"pmids\": [\"2046674\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define the transcription factors driving the mRNA decrease\", \"Limited to myogenic differentiation context\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Tested whether transcription-coupled repair is lesion-general, showing N-methylpurine base-excision repair is NOT preferential in the active DHFR gene, delimiting the scope of transcription-coupled repair.\",\n      \"evidence\": \"Alkaline hydrolysis quantitation of AP sites at DHFR, flanking, and mitochondrial DNA in CHO cells\",\n      \"pmids\": [\"8222061\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result; single lab\", \"Does not address other base lesions or repair pathways\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Defined the cell-cycle architecture of the DHFR promoter, distinguishing constitutive Sp1 binding from a cell-cycle-regulated E2F species that activates transcription at G1-to-S.\",\n      \"evidence\": \"High-resolution genomic DNase I footprinting and gel mobility shift with sequence-specific probes across the cell cycle\",\n      \"pmids\": [\"8552092\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the specific E2F family member by isoform\", \"Footprinting in CHO amplified locus may not capture single-copy regulation\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Asked whether transcription resumes only after lesion repair, showing RNA polymerase II can bypass UV lesions in DHFR before they are removed.\",\n      \"evidence\": \"In vivo RNA labeling and nuclear run-on at three DHFR positions in UV-irradiated CHO cells\",\n      \"pmids\": [\"10190552\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Compared to published photolesion kinetics from a separate study rather than matched controls\", \"Mechanism of polymerase bypass not defined\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Characterized RIP60 as a zinc-finger protein binding the DHFR replication origin oribeta, looping DNA via defined zinc-finger hands, linking the locus to origin function.\",\n      \"evidence\": \"One-hybrid cloning, footprinting, gel shift, AFM, and origin-dependent replication assay\",\n      \"pmids\": [\"10606657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RIP60 replication enhancer activity is weak; physiological role at oribeta unproven\", \"Does not establish requirement for DHFR origin firing in vivo\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Mechanistically dissected DHFR promoter repression during cell-cycle withdrawal into two cooperating complexes (Sp1-pRb-HDAC1 and p130-E2F4-DP1), explaining how histone deacetylation and pocket proteins coordinate repression.\",\n      \"evidence\": \"Reciprocal Co-IP, TSA treatment, reporter assays, and GAL4 recruitment in CHOC400 cells\",\n      \"pmids\": [\"11158299\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not map chromatin changes at the endogenous single-copy promoter genome-wide\", \"Quantitative contribution of each complex to repression unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Addressed how the active site coordinates catalysis at the level of protein structure, showing ligand binding reorganizes the residue interaction network through the cofactor and that active-site residues have highest network centrality.\",\n      \"evidence\": \"Residue interaction network analysis and molecular dynamics of native and 19 circularly permuted DHFRs\",\n      \"pmids\": [\"17571919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Computational only; no in vitro validation of permuted variants\", \"Network predictions not tied to measured catalytic parameters\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established DHFR as the required intermediate for folic-acid-mediated endothelial protection, regenerating H4B to recouple eNOS and restore NO.\",\n      \"evidence\": \"HPLC-fluorescence DHFR activity assay, siRNA knockdown, methotrexate inhibition, and NO/superoxide measurements in endothelial cells and Ang II mice\",\n      \"pmids\": [\"19660467\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define how folic acid upregulates DHFR expression\", \"Tissue specificity of the H4B-regeneration role not delineated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Tested whether fast protein motions drive the chemical step, showing hydride transfer in the bacterial ortholog is mass-independent and uncoupled from femtosecond-picosecond motions.\",\n      \"evidence\": \"Heavy-enzyme (13C/15N/2H) kinetics, kinetic isotope effects, and transition path sampling in ecDHFR\",\n      \"pmids\": [\"26652185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Performed in E. coli ortholog; human enzyme not tested\", \"Does not exclude slower conformational coupling to product release\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified a conserved residue (Asp122) tuning catalytically productive flexibility, establishing a Goldilocks principle whereby both excess and deficient coupled motion impair hydride transfer.\",\n      \"evidence\": \"QM/MM, molecular dynamics, correlated-motion analysis, and kinetics of Asp122 mutants in ecDHFR\",\n      \"pmids\": [\"30040418\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Primarily in silico; mutant kinetics not validated in vitro in this study\", \"Human DHFR equivalent residue not directly tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected DHFR loss-of-function to human disease, showing a homozygous mutation reduces enzyme activity/protein and causes megaloblastic anemia and cerebral folate deficiency with gene-dosage effect.\",\n      \"evidence\": \"DHFR enzyme activity and FMTX-binding assays in patient lymphoblastoid cells, homozygosity mapping, and LC-MS/MS folate profiling\",\n      \"pmids\": [\"21310277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single mutation/family; allelic spectrum not defined\", \"Mechanism of selective protein destabilization not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated a role for DHFR-dependent one-carbon metabolism in cancer stem-like cell biology, showing inhibition reduces brain tumor-initiating cell self-renewal and tumorigenicity.\",\n      \"evidence\": \"Methotrexate and EphB ligand inhibition, sphere formation, and cerebral organoid glioma model with four BTIC lines\",\n      \"pmids\": [\"33545223\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Two inhibition approaches but no genetic DHFR knockout validation\", \"Downstream one-carbon metabolites mediating self-renewal not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Distinguished DHFR degradation from inhibition, showing MTX-based PROTACs degrade DHFR via proteasome/E3 ligase and yield distinct phenotypes from enzymatic inhibition.\",\n      \"evidence\": \"PROTAC synthesis, Western blot degradation, proteasome and E3-ligase dependence controls in cancer cell lines\",\n      \"pmids\": [\"37875111\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why degradation is less lethal than inhibition is unexplained\", \"In vivo efficacy and selectivity not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DHFR catalytic, transcriptional, and metabolic regulation integrate in human tissues—and whether the bacterial-ortholog flexibility/Goldilocks mechanisms hold for human DHFR—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hydride-transfer flexibility findings derive from E. coli ortholog, not validated in human DHFR\", \"No structural model of human DHFR regulatory complexes at the promoter\", \"Mechanism linking folic acid sensing to DHFR transcriptional/protein upregulation undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [4, 5, 6]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [4, 5, 12]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":8,"faith_total":8,"faith_pct":100.0}}