Affinage

SHLD3

Shieldin complex subunit 3 · UniProt Q6ZNX1

Length
250 aa
Mass
28.8 kDa
Annotated
2026-06-10
18 papers in source corpus 12 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

SHLD3 is the apical subunit of the shieldin complex (SHLD3-REV7-SHLD2-SHLD1), which acts downstream of 53BP1 and RIF1 to protect DNA double-strand break ends from nucleolytic resection, thereby promoting non-homologous end-joining and immunoglobulin class-switch recombination (PMID:30022168, PMID:30046110, PMID:30022158). SHLD3 recruits shieldin to break sites through a direct interaction between its eIF4E-like domain and the HEAT-repeat domain of RIF1, an interaction required for focus formation, class switching, and PARP inhibitor sensitivity (PMID:37306046). Through a bipartite, ladle-shaped REV7-binding domain comprising an N-terminal loop and a C-terminal αC-helix that engages the REV7 'safety belt', SHLD3 binds REV7 with low-nanomolar affinity and nucleates a closed-REV7/open-REV7 conformational dimer that organizes assembly of the full complex onto SHLD2 (PMID:31796627, PMID:32332881, PMID:33597306). The conserved FXPWFP motif of SHLD3 binds closed-REV7 and blocks REV1 engagement, segregating shieldin/NHEJ activity from REV1/Polζ translesion synthesis (PMID:32332881). Once assembled, shieldin recruits CST-Polα to mediate fill-in synthesis that limits resection (PMID:30022158, PMID:40178294), while TRIP13 ATPase-driven disassembly of the SHLD3-REV7 interface—facilitated by p31comet and antagonized by CHAMP1 competition for the REV7 seatbelt—extracts REV7 and shifts repair toward homologous recombination (PMID:33597306, PMID:33051298, PMID:36044844). Loss of SHLD3-containing shieldin causes hyper-resection, defective class switching, and restoration of HR that confers PARP inhibitor resistance in BRCA1-deficient cells (PMID:30022168, PMID:30046110, PMID:30022158).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 2018 High

    Established SHLD3 as a bona fide DSB-repair factor by defining it as a shieldin subunit recruited downstream of 53BP1-RIF1 to protect DNA ends and promote NHEJ.

    Evidence Co-IP, ChIP, and genetic knockouts with class-switch recombination and resection readouts in cells

    PMID:30022158 PMID:30022168 PMID:30046110

    Open questions at the time
    • Did not resolve the atomic basis of SHLD3 interactions within the complex
    • Mechanism of how end-protection is enforced was not defined
  2. 2018 High

    Showed that loss of SHLD3-containing shieldin has therapeutic relevance by restoring HR and conferring PARP inhibitor resistance in BRCA1-deficient cells.

    Evidence Genetic knockouts/knockdowns with NHEJ, CSR, HR, and PARP inhibitor sensitivity assays

    PMID:30022158 PMID:30022168 PMID:30046110

    Open questions at the time
    • Did not identify the downstream effector that physically limits resection
  3. 2018 High

    Identified the effector that executes end-protection, linking shieldin to CST-Polα fill-in synthesis at break sites.

    Evidence Co-IP, immunofluorescence localization, and depletion epistasis with resection/HR readouts

    PMID:30022158 PMID:40178294

    Open questions at the time
    • Direct SHLD3-CST contact versus indirect recruitment not delineated
    • Stoichiometry of CST recruitment unresolved
  4. 2018 High

    Defined context-specificity of the 53BP1 pathway by showing SHLD3-containing shieldin is required for REV7-dependent NHEJ/CSR but dispensable for interstrand cross-link repair.

    Evidence Genetic knockouts with CSR, ICL repair, and epistasis analysis

    PMID:30046110

    Open questions at the time
    • Molecular basis for the differential REV7 function across pathways not established
  5. 2019 High

    Resolved how SHLD3 physically engages REV7, defining a bipartite ladle-shaped RBD that achieves high-affinity binding via the REV7 safety belt.

    Evidence Crystal structures of REV7-SHLD3 RBD at 2.2–2.3 Å with mutagenesis and binding kinetics

    PMID:31796627

    Open questions at the time
    • Did not show how the binary interaction scales to the full complex
    • Functional consequence of RBD mutants in cells not fully mapped
  6. 2020 High

    Revealed the organizing principle of shieldin assembly: SHLD3 nucleates a closed/open REV7 conformational dimer onto which SHLD2 docks, a dimer required for NHEJ.

    Evidence Crystal structure of SHLD3-REV7-SHLD2 ternary complex with dimer-interface mutagenesis and NHEJ assays

    PMID:32332881

    Open questions at the time
    • Kinetics and rate-limiting steps of assembly not addressed
    • How the dimer is disassembled was not resolved here
  7. 2020 High

    Explained how shieldin/NHEJ is insulated from translesion synthesis, showing the SHLD3 FXPWFP motif occupies closed-REV7 and blocks REV1.

    Evidence Crystal structure analysis with in vitro competition/binding assays and mutagenesis

    PMID:32332881

    Open questions at the time
    • In vivo competition dynamics between REV1 and SHLD3 not quantified
  8. 2020 Medium

    Identified the disassembly mechanism that switches repair toward HR: TRIP13 remodels REV7 to dissociate it from SHLD3, with p31comet bridging TRIP13-REV7 and driving PARPi resistance.

    Evidence Co-IP, chromatin fractionation, and HR/PARP inhibitor resistance assays

    PMID:33051298

    Open questions at the time
    • Single lab without structural validation of the remodeling intermediate
    • Quantitative contribution of p31comet versus TRIP13 alone unclear
  9. 2021 High

    Provided structural mechanism for TRIP13-driven disassembly, showing it threads the REV7 N-terminus into its channel and pulls the unfolded safety belt via ATP hydrolysis.

    Evidence Crystal and cryo-EM structures of binary/ternary and TRIP13-bound complexes with ATPase and disassembly assays

    PMID:33597306

    Open questions at the time
    • Cellular regulation of TRIP13 engagement timing not defined
  10. 2021 High

    Linked REV7 dimerization to assembly kinetics and pathway-choice regulation, showing SHLD2-mediated dimerization accelerates the MAD2L2-SHLD3 interaction and enables TRIP13-driven disassembly.

    Evidence Dimerization-defective MAD2L2 mutants, Co-IP, NHEJ and PARP inhibitor sensitivity assays

    PMID:34521823

    Open questions at the time
    • Upstream signals controlling the assembly/disassembly balance not identified
  11. 2022 Medium

    Identified CHAMP1 as a competitive antagonist that vacates the SHLD3-binding interface on REV7, lowering shieldin levels and promoting HR.

    Evidence Direct binding assays, Co-IP, resection and HR reporter assays

    PMID:36044844

    Open questions at the time
    • Single lab; physiological conditions favoring CHAMP1 over SHLD3 not defined
    • Interplay with TRIP13-mediated disassembly unresolved
  12. 2023 High

    Defined the recruitment receptor for shieldin, demonstrating a direct RIF1 HEAT-repeat / SHLD3 eIF4E-like domain interaction essential for focus formation, CSR, and PARPi sensitivity.

    Evidence AlphaFold2-Multimer prediction validated by in vitro pulldown and multiple cellular functional assays

    PMID:37306046

    Open questions at the time
    • High-resolution experimental structure of the RIF1-SHLD3 interface not determined
    • Regulation of this contact during the cell cycle not addressed
  13. 2023 Medium

    Established the kinetic logic of assembly through reconstitution, showing SHLD3-REV7 binding is the slow rate-limiting step and identifying a promiscuous SHLD3 DNA-binding domain.

    Evidence In vitro biochemical reconstitution, kinetic binding measurements, and DNA binding assays

    PMID:37031298

    Open questions at the time
    • Single lab; functional role of SHLD3 DNA binding in cells not demonstrated
    • Sequence/structure determinants of DNA binding specificity not defined
  14. 2025 Medium

    Refined the role of the shieldin-CST axis in immunity, showing CTC1 and SHLD1 are epistatic in preventing exacerbated resection but that the direct SHLD1-CTC1 motif is dispensable for class switching.

    Evidence CRISPR knockout B-cell lines with CSR, resection, chromosome break analysis and mutant complementation

    PMID:40178294

    Open questions at the time
    • Mechanism by which CST acts on resection independent of direct SHLD1 binding unresolved
    • SHLD3-specific contribution to this axis not isolated

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the assembly/disassembly equilibrium of SHLD3-nucleated shieldin is governed in vivo across the cell cycle and in response to DNA-damage signaling remains unresolved.
  • No defined upstream signal triggering TRIP13 or CHAMP1 antagonism of SHLD3-REV7
  • Functional significance of the SHLD3 DNA-binding domain in cells undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 3 GO:0003677 DNA binding 1 GO:0098772 molecular function regulator activity 1
Localization
GO:0000228 nuclear chromosome 2 GO:0005634 nucleus 1
Pathway
R-HSA-73894 DNA Repair 3 R-HSA-168256 Immune System 2
Partners
CHAMP1CTC1REV7RIF1SHLD2TRIP13
Complex memberships
shieldin (SHLD3-REV7-SHLD2-SHLD1)

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2018 SHLD3 (CTC-534A2.2) 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, protecting DNA ends from nucleolytic resection to promote non-homologous end-joining and immunoglobulin class-switch recombination. Co-immunoprecipitation, chromatin immunoprecipitation, genetic knockouts/knockdowns with CSR and resection readouts Nature High 30022158 30022168 30046110
2018 Loss of shieldin (including SHLD3) impairs NHEJ, causes hyper-resection of DSB ends, leads to defective immunoglobulin class switching, and causes resistance to PARP inhibition in BRCA1-deficient cells by restoring homologous recombination. Genetic knockouts and knockdowns with NHEJ efficiency assays, class-switch recombination assays, HR assays, and PARP inhibitor sensitivity measurements Nature High 30022158 30022168 30046110
2018 Shieldin complex (including SHLD3) interacts with and recruits CST (CTC1-STN1-TEN1), which localizes with Polymerase-α to sites of DNA damage in a 53BP1- and shieldin-dependent manner, mediating fill-in synthesis to control DSB end resection. Co-immunoprecipitation, immunofluorescence localization, epistasis by depletion with resection and HR readouts Nature High 30022158 40178294
2019 SHLD3 interacts with REV7 through a 'ladle-shaped' REV7-binding domain (RBD) comprising an N-terminal loop and a C-terminal α-helix (αC-helix); both elements are required for high-affinity (low-nanomolar) REV7 binding, and the REV7 'safety belt' region is essential for retaining the SHLD3 RBD. Crystal structures of REV7-SHLD3 RBD complex at 2.2–2.3 Å resolution, in vitro and in vivo binding analyses, mutagenesis of the αC-helix and N-terminal loop The Journal of biological chemistry High 31796627
2020 Crystal structure of the SHLD3-REV7-SHLD2 ternary complex reveals that SHLD3 mediates an unexpected closed (C)-REV7 / open (O)-REV7 conformational dimer; SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 via a β-sheet sandwich; disruption of the REV7 conformational dimer abolishes shieldin assembly and impairs NHEJ. Crystal structure determination, mutagenesis of the REV7 dimerization interface, NHEJ efficiency assays Nature communications High 32332881
2020 The conserved FXPWFP motif of SHLD3 binds to closed (C)-REV7 and blocks its binding to REV1, thereby excluding shieldin from the REV1/Pol ζ translesion synthesis complex and separating NHEJ from TLS functions. Crystal structure analysis and in vitro competition/binding assays with mutagenesis Nature communications High 32332881
2021 Crystal and cryo-EM structures of SHLD3-REV7 binary and SHLD2-SHLD3-REV7 ternary complexes confirm C-REV7/O-REV7 conformational heterodimerization induced by SHLD3; TRIP13 ATPase disassembles shieldin by inserting the N-terminus of REV7 into its central channel and pulling the unfolded C-REV7 safety-belt through ATP hydrolysis-driven rotatory motions. Crystal structure determination (SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary), cryo-EM of SHLD2-SHLD3-REV7-TRIP13 complex, ATPase and disassembly assays Proceedings of the National Academy of Sciences of the United States of America High 33597306
2021 MAD2L2 (REV7) dimerization mediated by SHLD2 accelerates the MAD2L2-SHLD3 interaction and is required for proper shieldin assembly; the presence of SHLD3 together with MAD2L2 dimerization enables shieldin to interact with the TRIP13 ATPase, which drives shieldin disassembly and regulates DNA repair pathway choice. Dimerization-defective MAD2L2 mutants, Co-immunoprecipitation, NHEJ assays, PARP inhibitor sensitivity assays Nature communications High 34521823
2020 TRIP13 promotes homologous recombination by remodeling REV7, causing its dissociation from the shieldin subunit SHLD3; p31comet facilitates this process by mediating the TRIP13-REV7 interaction and promoting extraction of REV7 from chromatin, thereby causing PARP inhibitor resistance. Co-immunoprecipitation, chromatin fractionation, HR and PARP inhibitor resistance assays Proceedings of the National Academy of Sciences of the United States of America Medium 33051298
2022 CHAMP1 binds directly to the REV7 seatbelt domain (the same interface used by SHLD3) and competes with SHLD3 binding, reducing shieldin complex levels and increasing DSB end resection to promote homologous recombination. Direct binding assays, Co-immunoprecipitation, resection assays, HR reporter assays Cell reports Medium 36044844
2023 AlphaFold2-Multimer predicted and in vitro pulldown plus cellular assays confirmed a direct physical interaction between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3; this RIF1-SHLD3 interaction is essential for shieldin recruitment to DSB sites and for antibody class switch recombination and PARP inhibitor sensitivity. AlphaFold2-Multimer structural prediction, in vitro pulldown assays, cellular co-immunoprecipitation, shieldin recruitment (focus formation) assays, CSR assays, PARP inhibitor sensitivity assays EMBO reports High 37306046
2023 SHLD3 contains a promiscuous DNA-binding domain; its interaction with the first REV7 molecule is remarkably slow (rate-limiting step in shieldin assembly), whereas the subsequent interaction with SHLD2 and the second REV7 molecule is fast and does not require structural remodeling. In vitro biochemical reconstitution, kinetic binding measurements, DNA binding assays Communications biology Medium 37031298
2018 Shieldin is essential for REV7-dependent DNA end-protection and NHEJ during class-switch recombination but is dispensable for REV7-dependent interstrand cross-link repair, demonstrating that SHLD3-containing shieldin explains the context-specificity of the 53BP1 pathway. Genetic knockouts, class-switch recombination assays, interstrand crosslink repair assays, epistasis analysis Nature High 30046110
2025 CTC1 (CST complex) and SHLD1 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 class switching, demonstrating that the direct SHLD1-CTC1 interaction through this motif is dispensable for promoting CSR. CRISPR knockout B-cell lines, CSR assays, resection assays, chromosome break/translocation analysis, complementation with SHLD1 mutant European journal of immunology Medium 40178294

Source papers

Stage 0 corpus · 18 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 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
2020 Molecular basis for assembly of the shieldin complex and its implications for NHEJ. Nature communications 41 32332881
2021 MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair. Nature communications 34 34521823
2020 p31comet promotes homologous recombination by inactivating REV7 through the TRIP13 ATPase. Proceedings of the National Academy of Sciences of the United States of America 30 33051298
2022 CHAMP1 binds to REV7/FANCV and promotes homologous recombination repair. Cell reports 26 36044844
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
2023 An AlphaFold2 map of the 53BP1 pathway identifies a direct SHLD3-RIF1 interaction critical for shieldin activity. EMBO reports 21 37306046
2020 SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus. EMBO reports 21 32558186
2019 Structural basis for shieldin complex subunit 3-mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair. The Journal of biological chemistry 20 31796627
2021 Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ. The Journal of biological chemistry 10 34174285
2020 CDH1 binds MAD2L2 in a Rev1-like pattern. Biochemical and biophysical research communications 7 32811646
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
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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