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SHLD2

Shieldin complex subunit 2 · UniProt Q86V20

Length
835 aa
Mass
93.7 kDa
Annotated
2026-06-10
28 papers in source corpus 17 papers cited in narrative 17 extracted findings
Cross-family judge vs UniProt: tie faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 2018 High

    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

    PMID:29789392 PMID:30022119 PMID:30022158 PMID:30022168 PMID:30046110 PMID:30154076 PMID:30254264

    Open questions at the time
    • Precise OB-fold residues contacting ssDNA versus protein partners not fully mapped
    • Mechanism by which ssDNA binding mechanically blocks nucleases not resolved
  2. 2018 High

    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

    PMID:30022168 PMID:30046110

    Open questions at the time
    • Why shieldin is dispensable for REV7-dependent ICL repair but essential for CSR not mechanistically explained at the molecular level
  3. 2018 High

    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

    PMID:30022158

    Open questions at the time
    • Direct molecular interface between SHLD2 and CST not defined
    • Order of fill-in relative to end-protection not temporally resolved
  4. 2020 High

    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

    PMID:32332881

    Open questions at the time
    • Structure of full complex bound to ssDNA not determined
    • How assembly is nucleated at the break in cells not shown
  5. 2020 High

    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

    PMID:32558186

    Open questions at the time
    • Basis for selectivity between CSR and V(D)J recombination not established
  6. 2021 High

    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

    PMID:33597306 PMID:34521823

    Open questions at the time
    • Spatiotemporal regulation of TRIP13-mediated disassembly at breaks unclear
    • Co-IP-based REV7 dimerization findings from single lab for the kinetic acceleration claim
  7. 2021 High

    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

    PMID:34354233

    Open questions at the time
    • How ASTE1 cleavage is coordinated with CST-Polα fill-in not resolved
    • Direct SHLD2-ASTE1 contact not demonstrated
  8. 2023 Medium

    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

    PMID:37031298

    Open questions at the time
    • Whether kinetic control operates the same way in cells not tested
    • Single-lab in vitro reconstitution
  9. 2022 Medium

    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

    PMID:36442094

    Open questions at the time
    • Single lab; reciprocal validation of the SHLD2-CCAR2 interaction limited
    • Mechanism by which CCAR2 restricts resection not defined
  10. 2024 Medium

    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

    PMID:38803048

    Open questions at the time
    • The E3 ligase writing the K64 ubiquitin mark on SHLD2 not identified
    • Single lab
  11. 2025 Medium

    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

    PMID:40178294

    Open questions at the time
    • Whether CST-shieldin coupling matters at other DSB contexts not addressed
    • Single lab
  12. 2025 Medium

    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)

    PMID:bio_10.1101_2025.07.04.662969

    Open questions at the time
    • Preprint, not yet peer-reviewed
    • Mechanistic basis of increased Polθ reliance in SHLD2-deficient cells not detailed

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003677 DNA binding 3 GO:0060090 molecular adaptor activity 2
Localization
GO:0000228 nuclear chromosome 2 GO:0005634 nucleus 2
Pathway
R-HSA-168256 Immune System 3 R-HSA-73894 DNA Repair 3
Partners
CCAR2REV7RIF1SHLD1SHLD3TRIP13USP25
Complex memberships
shieldin (SHLD1-SHLD2-SHLD3-REV7)

Evidence

Reading pass · 17 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2018 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. Co-immunoprecipitation, CRISPR screens, ssDNA binding assays, localization by immunofluorescence, epistasis with 53BP1/RIF1 pathway Nature High 30022119 30022158 30022168 30046110
2018 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. In vitro ssDNA binding assay, Co-IP, immunofluorescence recruitment assay, epistasis analysis Nature communications High 30254264
2018 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. Mass spectrometry-based interactome, NHEJ reporter assay, CSR assay, DNA end resection measurement, siRNA knockdown The EMBO journal High 30154076
2018 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. Protein domain analysis, Co-IP/pulldown with REV7, nuclear relocalization upon DNA damage by immunofluorescence, siRNA knockdown with drug sensitivity assays The EMBO journal Medium 29789392
2018 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. CRISPR knockout, NHEJ reporter assay, CSR assay, RPA/resection assays, PARP inhibitor resistance assays, mutagenesis of OB-fold domains Nature High 30022168
2018 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. Genetic epistasis in mouse models, CSR assay, ICL repair assay, PARP inhibitor sensitivity assay Nature High 30046110
2018 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. Co-immunoprecipitation, immunofluorescence co-localization, shieldin-dependent recruitment assay, RAD51 loading assay, PARP inhibitor sensitivity assay Nature High 30022158
2020 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. X-ray crystallography, mutagenesis of REV7 dimer interface, NHEJ efficiency assay, Co-IP Nature communications High 32332881
2021 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. X-ray crystallography, cryo-EM, ATPase activity assays, mutagenesis Proceedings of the National Academy of Sciences of the United States of America High 33597306
2020 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. Shld2 knockout mouse model, CSR assay in primary B cells, V(D)J recombination assay, embryonic lethality rescue assay EMBO reports High 32558186
2021 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. Co-immunoprecipitation, mutagenesis of MAD2L2 dimer interface, NHEJ reporter assay, TRIP13 interaction assay Nature communications Medium 34521823
2021 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. Nuclease activity assay, immunofluorescence with SHLD2-dependent localization, CRISPR knockout, NHEJ reporter, CSR assay, PARP inhibitor resistance assay Nature cell biology High 34354233
2022 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. Co-immunoprecipitation, CRISPR knockout epistasis, DSB end-resection assay, RAD51 loading assay, PARP inhibitor sensitivity assay, complementation with SHLD2 KO Proceedings of the National Academy of Sciences of the United States of America Medium 36442094
2023 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. In vitro binding kinetics assays, domain mapping, reconstitution of shieldin assembly intermediates Communications biology Medium 37031298
2024 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. Deubiquitination assay, site-directed mutagenesis (K64), Co-IP, NHEJ reporter assay, CSR assay in USP25 KO mice, PDX model with inhibitory peptide Advanced science (Weinheim, Baden-Wurttemberg, Germany) Medium 38803048
2025 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. CRISPR knockout screen, in vitro radiosensitization assay, in vivo xenograft model, chromosomal instability assay bioRxivpreprint Medium bio_10.1101_2025.07.04.662969
2025 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. Genetic epistasis with CTC1 and SHLD1 double knockout B cells, CSR assay, end-resection measurement, chromosomal translocation analysis, complementation with SHLD1 mutant European journal of immunology Medium 40178294

Source papers

Stage 0 corpus · 28 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2018 The shieldin complex mediates 53BP1-dependent DNA repair. Nature 505 30022168
2018 53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in. Nature 356 30022158
2018 Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells. Nature cell biology 338 30022119
2018 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ. Nature 254 30046110
2019 Shieldin - the protector of DNA ends. EMBO reports 188 30948458
2018 SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice. The EMBO journal 124 30154076
2016 GWAS of clinically defined gout and subtypes identifies multiple susceptibility loci that include urate transporter genes. Annals of the rheumatic diseases 108 27899376
2018 FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells. The EMBO journal 83 29789392
2018 An OB-fold complex controls the repair pathways for DNA double-strand breaks. Nature communications 73 30254264
2020 Molecular basis for assembly of the shieldin complex and its implications for NHEJ. Nature communications 41 32332881
2021 ASTE1 promotes shieldin-complex-mediated DNA repair by attenuating end resection. Nature cell biology 40 34354233
2021 MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair. Nature communications 34 34521823
2021 Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex. Proceedings of the National Academy of Sciences of the United States of America 22 33597306
2020 SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus. EMBO reports 21 32558186
2024 USP25 Elevates SHLD2-Mediated DNA Double-Strand Break Repair and Regulates Chemoresponse in Cancer. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 12 38803048
2022 CCAR2 functions downstream of the Shieldin complex to promote double-strand break end-joining. Proceedings of the National Academy of Sciences of the United States of America 12 36442094
2022 Identification of biomarkers of response to preoperative talazoparib monotherapy in treatment naïve gBRCA+ breast cancers. NPJ breast cancer 9 35538088
2020 CDH1 binds MAD2L2 in a Rev1-like pattern. Biochemical and biophysical research communications 7 32811646
2018 Genetic association and functional analysis of rs7903456 in FAM35A gene and hyperuricemia: a population based study. Scientific reports 7 29942023
2023 Prediction of CHO cell line stability using expression of DNA repair genes. Biotechnology journal 6 37970758
2024 Saccharomyces cerevisiae Rev7 promotes non-homologous end-joining by blocking Mre11 nuclease and Rad50's ATPase activities and homologous recombination. eLife 5 39630591
2023 Shieldin complex assembly kinetics and DNA binding by SHLD3. Communications biology 5 37031298
2023 Small-scale mutations are infrequent as mechanisms of resistance in post-PARP inhibitor tumour samples in high grade serous ovarian cancer. Scientific reports 5 38072854
2023 Single-nucleotide polymorphisms link gout with health-related lifestyle factors in Korean cohorts. PloS one 2 38060535
2020 FAM35A/SHLD2/RINN2: A novel determinant of double strand break repair pathway choice and genome stability in cancer. Environmental and molecular mutagenesis 2 32306447
2026 Resistance to neoadjuvant talazoparib in triple-negative breast cancer by BRN2-induced ATR/STAT3 pathways or SHLD2 subclone expansion. Proceedings of the National Academy of Sciences of the United States of America 1 41961819
2024 Naive and regulatory B-cell transcription patterns guide the increased risk of papillary thyroid carcinoma in obesity. Cellular and molecular biology (Noisy-le-Grand, France) 1 38678617
2025 CST Is Epistatic With Shieldin to Limit DNA Double-Strand Break End Resection and Promote Repair During Igh Class Switch Recombination. European journal of immunology 0 40178294

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