{"gene":"SHLD2","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":2018,"finding":"SHLD2 (FAM35A) is a subunit of the shieldin complex (SHLD1-SHLD2-SHLD3-REV7) that localizes to DNA double-strand break sites in a 53BP1- and RIF1-dependent manner, and its OB-fold domains bind single-stranded DNA to protect DNA ends from nucleolytic resection.","method":"Co-immunoprecipitation, CRISPR screens, ssDNA binding assays, localization by immunofluorescence, epistasis with 53BP1/RIF1 pathway","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — replicated across four independent labs in the same year using multiple orthogonal methods (Co-IP, in vitro ssDNA binding, genetic epistasis, CRISPR screens, functional rescue assays)","pmids":["30022168","30022158","30022119","30046110"],"is_preprint":false},{"year":2018,"finding":"SHLD2 accumulates at DSBs as part of a complex with REV7 and C20ORF196 (SHLD1) downstream of RIF1, and FAM35A preferentially binds single-strand DNA in vitro; epistasis analysis places both FAM35A and C20ORF196 in the same pathway as RIF1 for NHEJ.","method":"In vitro ssDNA binding assay, Co-IP, immunofluorescence recruitment assay, epistasis analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical assay plus epistasis analysis plus localization, replicating findings from concurrent papers","pmids":["30254264"],"is_preprint":false},{"year":2018,"finding":"SHLD2 interacts with REV7 and RIF1/53BP1, and FAM35A depletion impairs NHEJ-mediated DNA repair and class switch recombination in B cells, while antagonizing HR by limiting DNA end resection; FAM35A forms a complex with REV7 and SHLD1 identified by mass spectrometry.","method":"Mass spectrometry-based interactome, NHEJ reporter assay, CSR assay, DNA end resection measurement, siRNA knockdown","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS-based interaction screen plus multiple functional assays (NHEJ reporter, CSR, resection), replicating findings from concurrent papers","pmids":["30154076"],"is_preprint":false},{"year":2018,"finding":"FAM35A (SHLD2) has three C-terminal OB-fold domains homologous to those of RPA1, and was identified as a novel interactor of REV7/RIF1/53BP1; knockdown caused sensitivity to DNA-damaging agents, and in BRCA1-mutant cells FAM35A depletion increased resistance to camptothecin.","method":"Protein domain analysis, Co-IP/pulldown with REV7, nuclear relocalization upon DNA damage by immunofluorescence, siRNA knockdown with drug sensitivity assays","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reciprocal Co-IP plus functional knockdown phenotypes, single lab, multiple methods","pmids":["29789392"],"is_preprint":false},{"year":2018,"finding":"Loss of shieldin (including SHLD2) impairs non-homologous end-joining, leads to defective immunoglobulin class switching, causes hyper-resection, and restores homologous recombination in BRCA1-deficient cells, conferring resistance to PARP inhibitors; ssDNA binding by SHLD2 is critical for shieldin function.","method":"CRISPR knockout, NHEJ reporter assay, CSR assay, RPA/resection assays, PARP inhibitor resistance assays, mutagenesis of OB-fold domains","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis of OB-fold domains combined with multiple functional readouts, replicated across labs","pmids":["30022168"],"is_preprint":false},{"year":2018,"finding":"Shieldin (including SHLD2) is essential for REV7-dependent DNA end-protection and NHEJ during class-switch recombination, is dispensable for REV7-dependent interstrand cross-link repair, but supports toxic NHEJ in BRCA1-deficient cells; this explains immunological differences between 53bp1- and Rev7-deficient mice.","method":"Genetic epistasis in mouse models, CSR assay, ICL repair assay, PARP inhibitor sensitivity assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in mouse models with multiple orthogonal functional readouts, replicated across labs","pmids":["30046110"],"is_preprint":false},{"year":2018,"finding":"The 53BP1-RIF1-shieldin pathway controls DSB repair through recruitment of the CST complex, which interacts with shieldin and localizes with Polα to DNA damage sites in a 53BP1- and shieldin-dependent manner to mediate fill-in synthesis; SHLD2 acts upstream of CST-Polα.","method":"Co-immunoprecipitation, immunofluorescence co-localization, shieldin-dependent recruitment assay, RAD51 loading assay, PARP inhibitor sensitivity assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus functional epistasis, replicated in multiple subsequent studies","pmids":["30022158"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of the SHLD3-REV7-SHLD2 ternary complex reveals that shieldin assembly requires a conformational C-REV7/O-REV7 dimer mediated by SHLD3; SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 via β-sheet sandwich formation; disruption of REV7 conformational dimer abolishes shieldin assembly and impairs NHEJ; the FXPWFP motif of SHLD3 on C-REV7 blocks REV1 binding, excluding shieldin from the TLS complex.","method":"X-ray crystallography, mutagenesis of REV7 dimer interface, NHEJ efficiency assay, Co-IP","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus functional NHEJ assay in a single rigorous study","pmids":["32332881"],"is_preprint":false},{"year":2021,"finding":"Crystal structures of SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes show that shieldin assembly requires SHLD2-SHLD3-induced conformational heterodimerization of O-REV7 and C-REV7; cryo-EM structures of the SHLD2-SHLD3-REV7-TRIP13 complex reveal that TRIP13 disassembles shieldin by pulling the N-terminal peptide of C-REV7 through its central hexameric channel via ATP hydrolysis.","method":"X-ray crystallography, cryo-EM, ATPase activity assays, mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — orthogonal structural methods (crystal + cryo-EM) with mutagenesis and functional validation in one study","pmids":["33597306"],"is_preprint":false},{"year":2020,"finding":"SHLD2-deficient mice have defective class switch recombination in vivo, and SHLD2 loss suppresses embryonic lethality of the Brca1Δ11 mutation; lymphocyte development and RAG1/2-mediated V(D)J recombination are unaffected; SHLD2 prevents large deletions/loss of coding exons within the Igh locus during CSR.","method":"Shld2 knockout mouse model, CSR assay in primary B cells, V(D)J recombination assay, embryonic lethality rescue assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout mouse model with multiple orthogonal functional readouts","pmids":["32558186"],"is_preprint":false},{"year":2021,"finding":"MAD2L2 (REV7) dimerization is mediated by SHLD2 and accelerates MAD2L2-SHLD3 interaction; dimerization-defective MAD2L2 impairs shieldin assembly and NHEJ; MAD2L2 dimerization along with SHLD3 allows shieldin to interact with the TRIP13 ATPase, and appropriate TRIP13 levels are required for proper shieldin (dis)assembly and DNA repair activity.","method":"Co-immunoprecipitation, mutagenesis of MAD2L2 dimer interface, NHEJ reporter assay, TRIP13 interaction assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus mutagenesis plus NHEJ reporter, single lab","pmids":["34521823"],"is_preprint":false},{"year":2021,"finding":"ASTE1 is a structure-specific DNA endonuclease that specifically cleaves single-stranded and 3' overhang DNA; ASTE1 localizes to DNA damage sites in a shieldin-dependent manner (downstream of SHLD2); loss of ASTE1 impairs NHEJ, causes hyper-resection, and defective CSR, phenocopying loss of shieldin.","method":"Nuclease activity assay, immunofluorescence with SHLD2-dependent localization, CRISPR knockout, NHEJ reporter, CSR assay, PARP inhibitor resistance assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro nuclease assay plus genetic epistasis plus multiple functional readouts placing ASTE1 downstream of SHLD2","pmids":["34354233"],"is_preprint":false},{"year":2022,"finding":"CCAR2/DBC1 co-immunoprecipitates with the shieldin complex via its S1-like RNA-binding domain; CCAR2 functions downstream of shieldin to restrict DSB end-resection, and CCAR2 knockout is epistatic with knockout of shieldin proteins; FHA-domain-dependent targeting of CCAR2 to DSBs re-sensitized BRCA1-/-SHLD2-/- cells to PARP inhibitors.","method":"Co-immunoprecipitation, CRISPR knockout epistasis, DSB end-resection assay, RAD51 loading assay, PARP inhibitor sensitivity assay, complementation with SHLD2 KO","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus epistasis plus functional rescue, single lab","pmids":["36442094"],"is_preprint":false},{"year":2023,"finding":"SHLD3 contains a promiscuous DNA-binding domain at its N-terminus; the interaction between SHLD3 and the first REV7 is remarkably slow (rate-limiting step), whereas the interaction between SHLD3 and SHLD2 with a second REV7 is fast and does not require structural remodeling, revealing kinetic control of shieldin assembly.","method":"In vitro binding kinetics assays, domain mapping, reconstitution of shieldin assembly intermediates","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstitution with kinetic measurements, single lab","pmids":["37031298"],"is_preprint":false},{"year":2024,"finding":"USP25 deubiquitinates SHLD2 at the K64 site, which enhances SHLD2 binding to REV7 and promotes NHEJ; TRIM25 is the E3 ubiquitin ligase responsible for USP25 degradation; USP25 deficiency impairs NHEJ and reduces CSR in USP25-deficient mice; a peptide disrupting USP25-SHLD2 interaction impairs NHEJ in PDX models.","method":"Deubiquitination assay, site-directed mutagenesis (K64), Co-IP, NHEJ reporter assay, CSR assay in USP25 KO mice, PDX model with inhibitory peptide","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro deubiquitination assay plus mutagenesis plus mouse model, single lab","pmids":["38803048"],"is_preprint":false},{"year":2025,"finding":"SHLD2 loss is a synthetic vulnerability to Polθ inhibition combined with radiotherapy; SHLD2-deficient cancer cells are more reliant on Polθ to prevent DSB accumulation and chromosomal instability; identified by CRISPR knockout screen; validated in vitro and in vivo.","method":"CRISPR knockout screen, in vitro radiosensitization assay, in vivo xenograft model, chromosomal instability assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus in vitro and in vivo validation, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.07.04.662969"],"is_preprint":true},{"year":2025,"finding":"CST (CTC1) and shieldin (including SHLD2) are epistatic in preventing exacerbated DNA end resection and genetic instability during class switch recombination; a SHLD1 mutant defective in CST binding (SHLD1ΔLDLP) is fully proficient for CSR, demonstrating that the SHLD1-CTC1 interaction through this motif is dispensable for CST and SHLD function in CSR.","method":"Genetic epistasis with CTC1 and SHLD1 double knockout B cells, CSR assay, end-resection measurement, chromosomal translocation analysis, complementation with SHLD1 mutant","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus complementation with separation-of-function mutant, single lab","pmids":["40178294"],"is_preprint":false}],"current_model":"SHLD2 (FAM35A) is a core subunit of the four-protein shieldin complex (SHLD1-SHLD2-SHLD3-REV7/MAD2L2) that acts as the downstream effector of the 53BP1-RIF1 pathway: its C-terminal OB-fold domains bind single-stranded DNA at resected DSB ends to block further nucleolytic resection, thereby promoting NHEJ and immunoglobulin class switch recombination while suppressing homologous recombination; its stability and REV7-binding are regulated by ubiquitination at K64 (written by an E3 ligase, erased by USP25); structurally, SHLD2 interacts with the open (O) conformer of REV7 within a C-REV7/O-REV7 heterodimer scaffolded by SHLD3, and this assembled complex recruits downstream effectors CST-Polα for fill-in synthesis and ASTE1 for 3' ssDNA cleavage; the AAA+ ATPase TRIP13 disassembles shieldin by unfolding C-REV7 through its central channel."},"narrative":{"mechanistic_narrative":"SHLD2 (FAM35A) is a core subunit of the four-protein shieldin complex (SHLD1-SHLD2-SHLD3-REV7) that functions as the downstream effector of the 53BP1-RIF1 pathway in DNA double-strand break repair [PMID:30022168, PMID:30022158, PMID:30022119, PMID:30046110, PMID:30154076]. Recruited to break sites in a 53BP1- and RIF1-dependent manner, SHLD2 uses its C-terminal RPA1-like OB-fold domains to bind single-stranded DNA at resected ends and block further nucleolytic resection, thereby channeling repair toward non-homologous end-joining and away from homologous recombination [PMID:30022168, PMID:30022158, PMID:30022119, PMID:30046110, PMID:29789392]. Through this end-protective activity SHLD2 is required for immunoglobulin class switch recombination and guards the Igh locus against large deletions, while being dispensable for RAG-mediated V(D)J recombination [PMID:32558186, PMID:30154076]. Within the complex, SHLD2 binds the open conformer of REV7 in a C-REV7/O-REV7 conformational heterodimer scaffolded by SHLD3, an assembly governed by kinetic control in which the slow SHLD3-first-REV7 step is rate-limiting and the SHLD2-mediated second REV7 engagement is fast [PMID:32332881, PMID:34521823, PMID:37031298]; the AAA+ ATPase TRIP13 reverses this by threading the C-REV7 N-terminus through its hexameric channel to disassemble shieldin [PMID:33597306]. Once assembled, SHLD2 acts upstream of effectors that complete end-protection and fill-in: the CST-Polα complex for fill-in synthesis [PMID:30022158], the structure-specific endonuclease ASTE1 for 3' ssDNA cleavage [PMID:34354233], and CCAR2/DBC1 to restrict resection [PMID:36442094]; SHLD2 stability and REV7 binding are tuned by deubiquitination at K64 by USP25 [PMID:38803048]. Clinically, loss of SHLD2 restores resection and HR in BRCA1-deficient cells and confers PARP-inhibitor resistance, defining shieldin status as a determinant of therapeutic response [PMID:30022168, PMID:30046110].","teleology":[{"year":2018,"claim":"Establishing the identity and core function of shieldin answered how the 53BP1-RIF1 pathway physically protects DNA ends, defining SHLD2 as an ssDNA-binding end-protection factor that promotes NHEJ and antagonizes resection/HR.","evidence":"Convergent Co-IP, CRISPR screens, in vitro ssDNA binding, immunofluorescence and genetic epistasis across multiple labs; mouse and NHEJ/CSR reporter assays","pmids":["30022168","30022158","30022119","30046110","30254264","30154076","29789392"],"confidence":"High","gaps":["Precise OB-fold residues contacting ssDNA versus protein partners not fully mapped","Mechanism by which ssDNA binding mechanically blocks nucleases not resolved"]},{"year":2018,"claim":"Mutagenesis of the OB-fold domains and in vivo BRCA1 epistasis distinguished SHLD2's essential ssDNA-binding activity and linked shieldin loss to PARP-inhibitor resistance and toxic NHEJ in BRCA1-deficient cells.","evidence":"OB-fold domain mutagenesis with NHEJ/CSR/resection readouts and PARPi resistance assays; mouse genetic epistasis comparing 53bp1- and Rev7-deficient phenotypes","pmids":["30022168","30046110"],"confidence":"High","gaps":["Why shieldin is dispensable for REV7-dependent ICL repair but essential for CSR not mechanistically explained at the molecular level"]},{"year":2018,"claim":"Placing CST-Polα downstream of shieldin answered how protected ends are processed, showing SHLD2 acts upstream of fill-in synthesis at break sites.","evidence":"Co-IP, shieldin-dependent CST/Polα co-localization, RAD51 loading and PARPi sensitivity assays","pmids":["30022158"],"confidence":"High","gaps":["Direct molecular interface between SHLD2 and CST not defined","Order of fill-in relative to end-protection not temporally resolved"]},{"year":2020,"claim":"The SHLD3-REV7-SHLD2 crystal structure explained how shieldin assembles, revealing a SHLD3-scaffolded C-REV7/O-REV7 conformational dimer with SHLD2 bound to O-REV7 and a mechanism excluding shieldin from the REV1/TLS complex.","evidence":"X-ray crystallography, REV7 dimer-interface mutagenesis, Co-IP and NHEJ assays","pmids":["32332881"],"confidence":"High","gaps":["Structure of full complex bound to ssDNA not determined","How assembly is nucleated at the break in cells not shown"]},{"year":2020,"claim":"Knockout mice defined SHLD2's physiological role, showing it is required for CSR and protects the Igh locus from large deletions while being dispensable for V(D)J recombination, and that its loss rescues Brca1Δ11 embryonic lethality.","evidence":"Shld2 knockout mouse, CSR and V(D)J assays in primary B cells, embryonic lethality rescue","pmids":["32558186"],"confidence":"High","gaps":["Basis for selectivity between CSR and V(D)J recombination not established"]},{"year":2021,"claim":"Structural and biochemical work on REV7 dimerization and TRIP13 revealed the assembly/disassembly cycle, showing SHLD2 promotes REV7 conformational heterodimerization and that TRIP13 actively unfolds C-REV7 to dismantle shieldin.","evidence":"Crystallography, cryo-EM of SHLD2-SHLD3-REV7-TRIP13, ATPase assays, dimer-interface mutagenesis and NHEJ reporters","pmids":["33597306","34521823"],"confidence":"High","gaps":["Spatiotemporal regulation of TRIP13-mediated disassembly at breaks unclear","Co-IP-based REV7 dimerization findings from single lab for the kinetic acceleration claim"]},{"year":2021,"claim":"Identifying ASTE1 as a shieldin-dependent endonuclease placed a 3'-overhang-cleaving effector downstream of SHLD2, extending the end-processing arm of the pathway.","evidence":"In vitro nuclease assay, SHLD2-dependent localization, CRISPR knockout, NHEJ/CSR and PARPi resistance assays","pmids":["34354233"],"confidence":"High","gaps":["How ASTE1 cleavage is coordinated with CST-Polα fill-in not resolved","Direct SHLD2-ASTE1 contact not demonstrated"]},{"year":2023,"claim":"In vitro reconstitution defined the kinetic logic of assembly, identifying the slow SHLD3-first-REV7 step as rate-limiting versus the fast SHLD2-second-REV7 engagement.","evidence":"In vitro binding kinetics, domain mapping, reconstitution of assembly intermediates","pmids":["37031298"],"confidence":"Medium","gaps":["Whether kinetic control operates the same way in cells not tested","Single-lab in vitro reconstitution"]},{"year":2022,"claim":"CCAR2/DBC1 was placed downstream of shieldin as a resection-restricting factor, and its FHA-dependent targeting re-sensitized BRCA1-/-SHLD2-/- cells to PARP inhibitors.","evidence":"Co-IP, CRISPR knockout epistasis, resection/RAD51 assays, PARPi sensitivity with SHLD2 KO complementation","pmids":["36442094"],"confidence":"Medium","gaps":["Single lab; reciprocal validation of the SHLD2-CCAR2 interaction limited","Mechanism by which CCAR2 restricts resection not defined"]},{"year":2024,"claim":"Identifying USP25 as a SHLD2 deubiquitinase at K64 revealed post-translational control of shieldin, with K64 deubiquitination enhancing REV7 binding and NHEJ.","evidence":"Deubiquitination assay, K64 mutagenesis, Co-IP, NHEJ/CSR assays in USP25 KO mice, PDX with inhibitory peptide","pmids":["38803048"],"confidence":"Medium","gaps":["The E3 ligase writing the K64 ubiquitin mark on SHLD2 not identified","Single lab"]},{"year":2025,"claim":"CST-shieldin epistasis in CSR and a separation-of-function SHLD1 mutant clarified that the SHLD1-CTC1 motif interaction is dispensable for shieldin's role in class switch recombination.","evidence":"Genetic epistasis with CTC1/SHLD1 double-knockout B cells, CSR and resection assays, complementation with SHLD1ΔLDLP","pmids":["40178294"],"confidence":"Medium","gaps":["Whether CST-shieldin coupling matters at other DSB contexts not addressed","Single lab"]},{"year":2025,"claim":"A CRISPR screen identified SHLD2 loss as a synthetic vulnerability to Polθ inhibition plus radiotherapy, defining a therapeutic dependency in shieldin-deficient cancers.","evidence":"CRISPR knockout screen, in vitro radiosensitization, in vivo xenografts, chromosomal instability assays (preprint)","pmids":["bio_10.1101_2025.07.04.662969"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Mechanistic basis of increased Polθ reliance in SHLD2-deficient cells not detailed"]},{"year":null,"claim":"How SHLD2 OB-fold engagement of ssDNA is mechanically and temporally coordinated with downstream ASTE1 cleavage and CST-Polα fill-in, and what regulates shieldin assembly/disassembly cycling at individual breaks, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of SHLD2 bound to ssDNA at a resected end","Temporal ordering of end-protection, cleavage and fill-in not established","In vivo regulation of TRIP13-mediated disassembly unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,7]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,3]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,4,6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,5,9]}],"complexes":["shieldin (SHLD1-SHLD2-SHLD3-REV7)"],"partners":["SHLD1","SHLD3","REV7","RIF1","TRIP13","CCAR2","USP25"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86V20","full_name":"Shieldin complex subunit 2","aliases":["Protein FAM35A","RINN1-REV7-interacting novel NHEJ regulator 2","Shield complex subunit 2"],"length_aa":835,"mass_kda":93.7,"function":"Component of the shieldin complex, which plays an important role in repair of DNA double-stranded breaks (DSBs) (PubMed:29656893, PubMed:29789392). During G1 and S phase of the cell cycle, the complex functions downstream of TP53BP1 to promote non-homologous end joining (NHEJ) and suppress DNA end resection (PubMed:29656893, PubMed:29789392). Mediates various NHEJ-dependent processes including immunoglobulin class-switch recombination, and fusion of unprotected telomeres (PubMed:29656893)","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q86V20/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SHLD2","classification":"Not Classified","n_dependent_lines":160,"n_total_lines":1208,"dependency_fraction":0.13245033112582782},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SHLD2","total_profiled":1310},"omim":[{"mim_id":"620693","title":"ASTEROID HOMOLOG 1; ASTE1","url":"https://www.omim.org/entry/620693"},{"mim_id":"618030","title":"SHIELD COMPLEX, SUBUNIT 3; SHLD3","url":"https://www.omim.org/entry/618030"},{"mim_id":"618029","title":"SHIELD COMPLEX, SUBUNIT 2; SHLD2","url":"https://www.omim.org/entry/618029"},{"mim_id":"618028","title":"SHIELD COMPLEX, SUBUNIT 1; SHLD1","url":"https://www.omim.org/entry/618028"},{"mim_id":"605230","title":"TUMOR PROTEIN p53-BINDING PROTEIN 1; TP53BP1","url":"https://www.omim.org/entry/605230"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Actin filaments","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SHLD2"},"hgnc":{"alias_symbol":["MGC5560","bA163M19.1","FAM35A1","RINN2"],"prev_symbol":["FAM35A"]},"alphafold":{"accession":"Q86V20","domains":[{"cath_id":"2.40.50.140","chopping":"433-568","consensus_level":"high","plddt":84.3609,"start":433,"end":568},{"cath_id":"-","chopping":"658-695_732-824","consensus_level":"high","plddt":83.9853,"start":658,"end":824},{"cath_id":"2.40.10","chopping":"598-654","consensus_level":"high","plddt":63.3625,"start":598,"end":654}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86V20","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86V20-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86V20-F1-predicted_aligned_error_v6.png","plddt_mean":55.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SHLD2","jax_strain_url":"https://www.jax.org/strain/search?query=SHLD2"},"sequence":{"accession":"Q86V20","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86V20.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86V20/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86V20"}},"corpus_meta":[{"pmid":"30022168","id":"PMC_30022168","title":"The shieldin complex mediates 53BP1-dependent DNA repair.","date":"2018","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/30022168","citation_count":505,"is_preprint":false},{"pmid":"30022158","id":"PMC_30022158","title":"53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in.","date":"2018","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/30022158","citation_count":356,"is_preprint":false},{"pmid":"30022119","id":"PMC_30022119","title":"Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells.","date":"2018","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30022119","citation_count":338,"is_preprint":false},{"pmid":"30046110","id":"PMC_30046110","title":"53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ.","date":"2018","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/30046110","citation_count":254,"is_preprint":false},{"pmid":"30948458","id":"PMC_30948458","title":"Shieldin - the protector of DNA ends.","date":"2019","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/30948458","citation_count":188,"is_preprint":false},{"pmid":"30154076","id":"PMC_30154076","title":"SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice.","date":"2018","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/30154076","citation_count":124,"is_preprint":false},{"pmid":"27899376","id":"PMC_27899376","title":"GWAS of clinically defined gout and subtypes identifies multiple susceptibility loci that include urate transporter genes.","date":"2016","source":"Annals of the rheumatic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/27899376","citation_count":108,"is_preprint":false},{"pmid":"29789392","id":"PMC_29789392","title":"FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells.","date":"2018","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/29789392","citation_count":83,"is_preprint":false},{"pmid":"30254264","id":"PMC_30254264","title":"An OB-fold complex controls the repair pathways for DNA double-strand breaks.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/30254264","citation_count":73,"is_preprint":false},{"pmid":"32332881","id":"PMC_32332881","title":"Molecular basis for assembly of the shieldin complex and its implications for NHEJ.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32332881","citation_count":41,"is_preprint":false},{"pmid":"34354233","id":"PMC_34354233","title":"ASTE1 promotes shieldin-complex-mediated DNA repair by attenuating end resection.","date":"2021","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/34354233","citation_count":40,"is_preprint":false},{"pmid":"34521823","id":"PMC_34521823","title":"MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34521823","citation_count":34,"is_preprint":false},{"pmid":"33597306","id":"PMC_33597306","title":"Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex.","date":"2021","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/33597306","citation_count":22,"is_preprint":false},{"pmid":"32558186","id":"PMC_32558186","title":"SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus.","date":"2020","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/32558186","citation_count":21,"is_preprint":false},{"pmid":"38803048","id":"PMC_38803048","title":"USP25 Elevates SHLD2-Mediated DNA Double-Strand Break Repair and Regulates Chemoresponse in Cancer.","date":"2024","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/38803048","citation_count":12,"is_preprint":false},{"pmid":"36442094","id":"PMC_36442094","title":"CCAR2 functions downstream of the Shieldin complex to promote double-strand break end-joining.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/36442094","citation_count":12,"is_preprint":false},{"pmid":"35538088","id":"PMC_35538088","title":"Identification of biomarkers of response to preoperative talazoparib monotherapy in treatment naïve gBRCA+ breast cancers.","date":"2022","source":"NPJ breast cancer","url":"https://pubmed.ncbi.nlm.nih.gov/35538088","citation_count":9,"is_preprint":false},{"pmid":"29942023","id":"PMC_29942023","title":"Genetic association and functional analysis of rs7903456 in FAM35A gene and hyperuricemia: a population based study.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29942023","citation_count":7,"is_preprint":false},{"pmid":"32811646","id":"PMC_32811646","title":"CDH1 binds MAD2L2 in a Rev1-like pattern.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/32811646","citation_count":7,"is_preprint":false},{"pmid":"37970758","id":"PMC_37970758","title":"Prediction of CHO cell line stability using expression of DNA repair genes.","date":"2023","source":"Biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/37970758","citation_count":6,"is_preprint":false},{"pmid":"37031298","id":"PMC_37031298","title":"Shieldin complex assembly kinetics and DNA binding by SHLD3.","date":"2023","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/37031298","citation_count":5,"is_preprint":false},{"pmid":"39630591","id":"PMC_39630591","title":"Saccharomyces cerevisiae Rev7 promotes non-homologous end-joining by blocking Mre11 nuclease and Rad50's ATPase activities and homologous recombination.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/39630591","citation_count":5,"is_preprint":false},{"pmid":"38072854","id":"PMC_38072854","title":"Small-scale mutations are infrequent as mechanisms of resistance in post-PARP inhibitor tumour samples in high grade serous ovarian cancer.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/38072854","citation_count":5,"is_preprint":false},{"pmid":"32306447","id":"PMC_32306447","title":"FAM35A/SHLD2/RINN2: A novel determinant of double strand break repair pathway choice and genome stability in cancer.","date":"2020","source":"Environmental and molecular mutagenesis","url":"https://pubmed.ncbi.nlm.nih.gov/32306447","citation_count":2,"is_preprint":false},{"pmid":"38060535","id":"PMC_38060535","title":"Single-nucleotide polymorphisms link gout with health-related lifestyle factors in Korean cohorts.","date":"2023","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/38060535","citation_count":2,"is_preprint":false},{"pmid":"41961819","id":"PMC_41961819","title":"Resistance to neoadjuvant talazoparib in triple-negative breast cancer by BRN2-induced ATR/STAT3 pathways or SHLD2 subclone expansion.","date":"2026","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41961819","citation_count":1,"is_preprint":false},{"pmid":"38678617","id":"PMC_38678617","title":"Naive and regulatory B-cell transcription patterns guide the increased risk of papillary thyroid carcinoma in obesity.","date":"2024","source":"Cellular and molecular biology (Noisy-le-Grand, France)","url":"https://pubmed.ncbi.nlm.nih.gov/38678617","citation_count":1,"is_preprint":false},{"pmid":"40178294","id":"PMC_40178294","title":"CST Is Epistatic With Shieldin to Limit DNA Double-Strand Break End Resection and Promote Repair During Igh Class Switch Recombination.","date":"2025","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40178294","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.04.662969","title":"SHLD2 loss is a synthetic vulnerability to Polθ inhibition combined with radiotherapy","date":"2025-07-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.04.662969","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15910,"output_tokens":4520,"usd":0.057765,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12468,"output_tokens":4333,"usd":0.085333,"stage2_stop_reason":"end_turn"},"total_usd":0.143098,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"SHLD2 (FAM35A) is a subunit of the shieldin complex (SHLD1-SHLD2-SHLD3-REV7) that localizes to DNA double-strand break sites in a 53BP1- and RIF1-dependent manner, and its OB-fold domains bind single-stranded DNA to protect DNA ends from nucleolytic resection.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR screens, ssDNA binding assays, localization by immunofluorescence, epistasis with 53BP1/RIF1 pathway\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — replicated across four independent labs in the same year using multiple orthogonal methods (Co-IP, in vitro ssDNA binding, genetic epistasis, CRISPR screens, functional rescue assays)\",\n      \"pmids\": [\"30022168\", \"30022158\", \"30022119\", \"30046110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SHLD2 accumulates at DSBs as part of a complex with REV7 and C20ORF196 (SHLD1) downstream of RIF1, and FAM35A preferentially binds single-strand DNA in vitro; epistasis analysis places both FAM35A and C20ORF196 in the same pathway as RIF1 for NHEJ.\",\n      \"method\": \"In vitro ssDNA binding assay, Co-IP, immunofluorescence recruitment assay, epistasis analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical assay plus epistasis analysis plus localization, replicating findings from concurrent papers\",\n      \"pmids\": [\"30254264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SHLD2 interacts with REV7 and RIF1/53BP1, and FAM35A depletion impairs NHEJ-mediated DNA repair and class switch recombination in B cells, while antagonizing HR by limiting DNA end resection; FAM35A forms a complex with REV7 and SHLD1 identified by mass spectrometry.\",\n      \"method\": \"Mass spectrometry-based interactome, NHEJ reporter assay, CSR assay, DNA end resection measurement, siRNA knockdown\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS-based interaction screen plus multiple functional assays (NHEJ reporter, CSR, resection), replicating findings from concurrent papers\",\n      \"pmids\": [\"30154076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"FAM35A (SHLD2) has three C-terminal OB-fold domains homologous to those of RPA1, and was identified as a novel interactor of REV7/RIF1/53BP1; knockdown caused sensitivity to DNA-damaging agents, and in BRCA1-mutant cells FAM35A depletion increased resistance to camptothecin.\",\n      \"method\": \"Protein domain analysis, Co-IP/pulldown with REV7, nuclear relocalization upon DNA damage by immunofluorescence, siRNA knockdown with drug sensitivity assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reciprocal Co-IP plus functional knockdown phenotypes, single lab, multiple methods\",\n      \"pmids\": [\"29789392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Loss of shieldin (including SHLD2) impairs non-homologous end-joining, leads to defective immunoglobulin class switching, causes hyper-resection, and restores homologous recombination in BRCA1-deficient cells, conferring resistance to PARP inhibitors; ssDNA binding by SHLD2 is critical for shieldin function.\",\n      \"method\": \"CRISPR knockout, NHEJ reporter assay, CSR assay, RPA/resection assays, PARP inhibitor resistance assays, mutagenesis of OB-fold domains\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis of OB-fold domains combined with multiple functional readouts, replicated across labs\",\n      \"pmids\": [\"30022168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Shieldin (including SHLD2) is essential for REV7-dependent DNA end-protection and NHEJ during class-switch recombination, is dispensable for REV7-dependent interstrand cross-link repair, but supports toxic NHEJ in BRCA1-deficient cells; this explains immunological differences between 53bp1- and Rev7-deficient mice.\",\n      \"method\": \"Genetic epistasis in mouse models, CSR assay, ICL repair assay, PARP inhibitor sensitivity assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in mouse models with multiple orthogonal functional readouts, replicated across labs\",\n      \"pmids\": [\"30046110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The 53BP1-RIF1-shieldin pathway controls DSB repair through recruitment of the CST complex, which interacts with shieldin and localizes with Polα to DNA damage sites in a 53BP1- and shieldin-dependent manner to mediate fill-in synthesis; SHLD2 acts upstream of CST-Polα.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, shieldin-dependent recruitment assay, RAD51 loading assay, PARP inhibitor sensitivity assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus functional epistasis, replicated in multiple subsequent studies\",\n      \"pmids\": [\"30022158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of the SHLD3-REV7-SHLD2 ternary complex reveals that shieldin assembly requires a conformational C-REV7/O-REV7 dimer mediated by SHLD3; SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 via β-sheet sandwich formation; disruption of REV7 conformational dimer abolishes shieldin assembly and impairs NHEJ; the FXPWFP motif of SHLD3 on C-REV7 blocks REV1 binding, excluding shieldin from the TLS complex.\",\n      \"method\": \"X-ray crystallography, mutagenesis of REV7 dimer interface, NHEJ efficiency assay, Co-IP\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus functional NHEJ assay in a single rigorous study\",\n      \"pmids\": [\"32332881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structures of SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes show that shieldin assembly requires SHLD2-SHLD3-induced conformational heterodimerization of O-REV7 and C-REV7; cryo-EM structures of the SHLD2-SHLD3-REV7-TRIP13 complex reveal that TRIP13 disassembles shieldin by pulling the N-terminal peptide of C-REV7 through its central hexameric channel via ATP hydrolysis.\",\n      \"method\": \"X-ray crystallography, cryo-EM, ATPase activity assays, mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — orthogonal structural methods (crystal + cryo-EM) with mutagenesis and functional validation in one study\",\n      \"pmids\": [\"33597306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SHLD2-deficient mice have defective class switch recombination in vivo, and SHLD2 loss suppresses embryonic lethality of the Brca1Δ11 mutation; lymphocyte development and RAG1/2-mediated V(D)J recombination are unaffected; SHLD2 prevents large deletions/loss of coding exons within the Igh locus during CSR.\",\n      \"method\": \"Shld2 knockout mouse model, CSR assay in primary B cells, V(D)J recombination assay, embryonic lethality rescue assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout mouse model with multiple orthogonal functional readouts\",\n      \"pmids\": [\"32558186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MAD2L2 (REV7) dimerization is mediated by SHLD2 and accelerates MAD2L2-SHLD3 interaction; dimerization-defective MAD2L2 impairs shieldin assembly and NHEJ; MAD2L2 dimerization along with SHLD3 allows shieldin to interact with the TRIP13 ATPase, and appropriate TRIP13 levels are required for proper shieldin (dis)assembly and DNA repair activity.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis of MAD2L2 dimer interface, NHEJ reporter assay, TRIP13 interaction assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus mutagenesis plus NHEJ reporter, single lab\",\n      \"pmids\": [\"34521823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ASTE1 is a structure-specific DNA endonuclease that specifically cleaves single-stranded and 3' overhang DNA; ASTE1 localizes to DNA damage sites in a shieldin-dependent manner (downstream of SHLD2); loss of ASTE1 impairs NHEJ, causes hyper-resection, and defective CSR, phenocopying loss of shieldin.\",\n      \"method\": \"Nuclease activity assay, immunofluorescence with SHLD2-dependent localization, CRISPR knockout, NHEJ reporter, CSR assay, PARP inhibitor resistance assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro nuclease assay plus genetic epistasis plus multiple functional readouts placing ASTE1 downstream of SHLD2\",\n      \"pmids\": [\"34354233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CCAR2/DBC1 co-immunoprecipitates with the shieldin complex via its S1-like RNA-binding domain; CCAR2 functions downstream of shieldin to restrict DSB end-resection, and CCAR2 knockout is epistatic with knockout of shieldin proteins; FHA-domain-dependent targeting of CCAR2 to DSBs re-sensitized BRCA1-/-SHLD2-/- cells to PARP inhibitors.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR knockout epistasis, DSB end-resection assay, RAD51 loading assay, PARP inhibitor sensitivity assay, complementation with SHLD2 KO\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus epistasis plus functional rescue, single lab\",\n      \"pmids\": [\"36442094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SHLD3 contains a promiscuous DNA-binding domain at its N-terminus; the interaction between SHLD3 and the first REV7 is remarkably slow (rate-limiting step), whereas the interaction between SHLD3 and SHLD2 with a second REV7 is fast and does not require structural remodeling, revealing kinetic control of shieldin assembly.\",\n      \"method\": \"In vitro binding kinetics assays, domain mapping, reconstitution of shieldin assembly intermediates\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstitution with kinetic measurements, single lab\",\n      \"pmids\": [\"37031298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP25 deubiquitinates SHLD2 at the K64 site, which enhances SHLD2 binding to REV7 and promotes NHEJ; TRIM25 is the E3 ubiquitin ligase responsible for USP25 degradation; USP25 deficiency impairs NHEJ and reduces CSR in USP25-deficient mice; a peptide disrupting USP25-SHLD2 interaction impairs NHEJ in PDX models.\",\n      \"method\": \"Deubiquitination assay, site-directed mutagenesis (K64), Co-IP, NHEJ reporter assay, CSR assay in USP25 KO mice, PDX model with inhibitory peptide\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro deubiquitination assay plus mutagenesis plus mouse model, single lab\",\n      \"pmids\": [\"38803048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SHLD2 loss is a synthetic vulnerability to Polθ inhibition combined with radiotherapy; SHLD2-deficient cancer cells are more reliant on Polθ to prevent DSB accumulation and chromosomal instability; identified by CRISPR knockout screen; validated in vitro and in vivo.\",\n      \"method\": \"CRISPR knockout screen, in vitro radiosensitization assay, in vivo xenograft model, chromosomal instability assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus in vitro and in vivo validation, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.07.04.662969\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CST (CTC1) and shieldin (including SHLD2) are epistatic in preventing exacerbated DNA end resection and genetic instability during class switch recombination; a SHLD1 mutant defective in CST binding (SHLD1ΔLDLP) is fully proficient for CSR, demonstrating that the SHLD1-CTC1 interaction through this motif is dispensable for CST and SHLD function in CSR.\",\n      \"method\": \"Genetic epistasis with CTC1 and SHLD1 double knockout B cells, CSR assay, end-resection measurement, chromosomal translocation analysis, complementation with SHLD1 mutant\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus complementation with separation-of-function mutant, single lab\",\n      \"pmids\": [\"40178294\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SHLD2 (FAM35A) is a core subunit of the four-protein shieldin complex (SHLD1-SHLD2-SHLD3-REV7/MAD2L2) that acts as the downstream effector of the 53BP1-RIF1 pathway: its C-terminal OB-fold domains bind single-stranded DNA at resected DSB ends to block further nucleolytic resection, thereby promoting NHEJ and immunoglobulin class switch recombination while suppressing homologous recombination; its stability and REV7-binding are regulated by ubiquitination at K64 (written by an E3 ligase, erased by USP25); structurally, SHLD2 interacts with the open (O) conformer of REV7 within a C-REV7/O-REV7 heterodimer scaffolded by SHLD3, and this assembled complex recruits downstream effectors CST-Polα for fill-in synthesis and ASTE1 for 3' ssDNA cleavage; the AAA+ ATPase TRIP13 disassembles shieldin by unfolding C-REV7 through its central channel.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SHLD2 (FAM35A) is a core subunit of the four-protein shieldin complex (SHLD1-SHLD2-SHLD3-REV7) that functions as the downstream effector of the 53BP1-RIF1 pathway in DNA double-strand break repair [#0, #2]. Recruited to break sites in a 53BP1- and RIF1-dependent manner, SHLD2 uses its C-terminal RPA1-like OB-fold domains to bind single-stranded DNA at resected ends and block further nucleolytic resection, thereby channeling repair toward non-homologous end-joining and away from homologous recombination [#0, #3, #4]. Through this end-protective activity SHLD2 is required for immunoglobulin class switch recombination and guards the Igh locus against large deletions, while being dispensable for RAG-mediated V(D)J recombination [#9, #2]. Within the complex, SHLD2 binds the open conformer of REV7 in a C-REV7/O-REV7 conformational heterodimer scaffolded by SHLD3, an assembly governed by kinetic control in which the slow SHLD3-first-REV7 step is rate-limiting and the SHLD2-mediated second REV7 engagement is fast [#7, #10, #13]; the AAA+ ATPase TRIP13 reverses this by threading the C-REV7 N-terminus through its hexameric channel to disassemble shieldin [#8]. Once assembled, SHLD2 acts upstream of effectors that complete end-protection and fill-in: the CST-Polα complex for fill-in synthesis [#6], the structure-specific endonuclease ASTE1 for 3' ssDNA cleavage [#11], and CCAR2/DBC1 to restrict resection [#12]; SHLD2 stability and REV7 binding are tuned by deubiquitination at K64 by USP25 [#14]. Clinically, loss of SHLD2 restores resection and HR in BRCA1-deficient cells and confers PARP-inhibitor resistance, defining shieldin status as a determinant of therapeutic response [#4, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Establishing the identity and core function of shieldin answered how the 53BP1-RIF1 pathway physically protects DNA ends, defining SHLD2 as an ssDNA-binding end-protection factor that promotes NHEJ and antagonizes resection/HR.\",\n      \"evidence\": \"Convergent Co-IP, CRISPR screens, in vitro ssDNA binding, immunofluorescence and genetic epistasis across multiple labs; mouse and NHEJ/CSR reporter assays\",\n      \"pmids\": [\"30022168\", \"30022158\", \"30022119\", \"30046110\", \"30254264\", \"30154076\", \"29789392\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise OB-fold residues contacting ssDNA versus protein partners not fully mapped\", \"Mechanism by which ssDNA binding mechanically blocks nucleases not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Mutagenesis of the OB-fold domains and in vivo BRCA1 epistasis distinguished SHLD2's essential ssDNA-binding activity and linked shieldin loss to PARP-inhibitor resistance and toxic NHEJ in BRCA1-deficient cells.\",\n      \"evidence\": \"OB-fold domain mutagenesis with NHEJ/CSR/resection readouts and PARPi resistance assays; mouse genetic epistasis comparing 53bp1- and Rev7-deficient phenotypes\",\n      \"pmids\": [\"30022168\", \"30046110\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why shieldin is dispensable for REV7-dependent ICL repair but essential for CSR not mechanistically explained at the molecular level\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placing CST-Polα downstream of shieldin answered how protected ends are processed, showing SHLD2 acts upstream of fill-in synthesis at break sites.\",\n      \"evidence\": \"Co-IP, shieldin-dependent CST/Polα co-localization, RAD51 loading and PARPi sensitivity assays\",\n      \"pmids\": [\"30022158\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular interface between SHLD2 and CST not defined\", \"Order of fill-in relative to end-protection not temporally resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The SHLD3-REV7-SHLD2 crystal structure explained how shieldin assembles, revealing a SHLD3-scaffolded C-REV7/O-REV7 conformational dimer with SHLD2 bound to O-REV7 and a mechanism excluding shieldin from the REV1/TLS complex.\",\n      \"evidence\": \"X-ray crystallography, REV7 dimer-interface mutagenesis, Co-IP and NHEJ assays\",\n      \"pmids\": [\"32332881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full complex bound to ssDNA not determined\", \"How assembly is nucleated at the break in cells not shown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Knockout mice defined SHLD2's physiological role, showing it is required for CSR and protects the Igh locus from large deletions while being dispensable for V(D)J recombination, and that its loss rescues Brca1Δ11 embryonic lethality.\",\n      \"evidence\": \"Shld2 knockout mouse, CSR and V(D)J assays in primary B cells, embryonic lethality rescue\",\n      \"pmids\": [\"32558186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis for selectivity between CSR and V(D)J recombination not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Structural and biochemical work on REV7 dimerization and TRIP13 revealed the assembly/disassembly cycle, showing SHLD2 promotes REV7 conformational heterodimerization and that TRIP13 actively unfolds C-REV7 to dismantle shieldin.\",\n      \"evidence\": \"Crystallography, cryo-EM of SHLD2-SHLD3-REV7-TRIP13, ATPase assays, dimer-interface mutagenesis and NHEJ reporters\",\n      \"pmids\": [\"33597306\", \"34521823\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatiotemporal regulation of TRIP13-mediated disassembly at breaks unclear\", \"Co-IP-based REV7 dimerization findings from single lab for the kinetic acceleration claim\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying ASTE1 as a shieldin-dependent endonuclease placed a 3'-overhang-cleaving effector downstream of SHLD2, extending the end-processing arm of the pathway.\",\n      \"evidence\": \"In vitro nuclease assay, SHLD2-dependent localization, CRISPR knockout, NHEJ/CSR and PARPi resistance assays\",\n      \"pmids\": [\"34354233\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ASTE1 cleavage is coordinated with CST-Polα fill-in not resolved\", \"Direct SHLD2-ASTE1 contact not demonstrated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"In vitro reconstitution defined the kinetic logic of assembly, identifying the slow SHLD3-first-REV7 step as rate-limiting versus the fast SHLD2-second-REV7 engagement.\",\n      \"evidence\": \"In vitro binding kinetics, domain mapping, reconstitution of assembly intermediates\",\n      \"pmids\": [\"37031298\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether kinetic control operates the same way in cells not tested\", \"Single-lab in vitro reconstitution\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"CCAR2/DBC1 was placed downstream of shieldin as a resection-restricting factor, and its FHA-dependent targeting re-sensitized BRCA1-/-SHLD2-/- cells to PARP inhibitors.\",\n      \"evidence\": \"Co-IP, CRISPR knockout epistasis, resection/RAD51 assays, PARPi sensitivity with SHLD2 KO complementation\",\n      \"pmids\": [\"36442094\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; reciprocal validation of the SHLD2-CCAR2 interaction limited\", \"Mechanism by which CCAR2 restricts resection not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying USP25 as a SHLD2 deubiquitinase at K64 revealed post-translational control of shieldin, with K64 deubiquitination enhancing REV7 binding and NHEJ.\",\n      \"evidence\": \"Deubiquitination assay, K64 mutagenesis, Co-IP, NHEJ/CSR assays in USP25 KO mice, PDX with inhibitory peptide\",\n      \"pmids\": [\"38803048\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The E3 ligase writing the K64 ubiquitin mark on SHLD2 not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"CST-shieldin epistasis in CSR and a separation-of-function SHLD1 mutant clarified that the SHLD1-CTC1 motif interaction is dispensable for shieldin's role in class switch recombination.\",\n      \"evidence\": \"Genetic epistasis with CTC1/SHLD1 double-knockout B cells, CSR and resection assays, complementation with SHLD1ΔLDLP\",\n      \"pmids\": [\"40178294\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CST-shieldin coupling matters at other DSB contexts not addressed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A CRISPR screen identified SHLD2 loss as a synthetic vulnerability to Polθ inhibition plus radiotherapy, defining a therapeutic dependency in shieldin-deficient cancers.\",\n      \"evidence\": \"CRISPR knockout screen, in vitro radiosensitization, in vivo xenografts, chromosomal instability assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.07.04.662969\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Mechanistic basis of increased Polθ reliance in SHLD2-deficient cells not detailed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SHLD2 OB-fold engagement of ssDNA is mechanically and temporally coordinated with downstream ASTE1 cleavage and CST-Polα fill-in, and what regulates shieldin assembly/disassembly cycling at individual breaks, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of SHLD2 bound to ssDNA at a resected end\", \"Temporal ordering of end-protection, cleavage and fill-in not established\", \"In vivo regulation of TRIP13-mediated disassembly unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 4, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 5, 9]}\n    ],\n    \"complexes\": [\"shieldin (SHLD1-SHLD2-SHLD3-REV7)\"],\n    \"partners\": [\"SHLD1\", \"SHLD3\", \"REV7\", \"RIF1\", \"TRIP13\", \"CCAR2\", \"USP25\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}