Affinage

SHLD3

Shieldin complex subunit 3 · UniProt Q6ZNX1

Length
250 aa
Mass
28.8 kDa
Annotated
2026-04-28
18 papers in source corpus 13 papers cited in narrative 12 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

SHLD3 is the apical scaffolding subunit of the shieldin complex (SHLD3–REV7–SHLD2–SHLD1) that connects the 53BP1–RIF1 DNA damage response axis to the protection of DNA double-strand break ends from nucleolytic resection, thereby promoting non-homologous end joining and immunoglobulin class-switch recombination (PMID:30022168, PMID:30022158). SHLD3 binds RIF1 through its eIF4E-like domain and engages REV7 through a bipartite REV7-binding domain (N-terminal loop plus αC-helix engaging the REV7 safety belt), with this initial association being kinetically slow and rate-limiting for shieldin assembly; subsequent SHLD2-assisted capture of a second REV7 molecule generates a closed–open REV7 conformational heterodimer that constitutes the assembled complex core (PMID:31796627, PMID:32332881, PMID:37031298, PMID:37306046). A conserved FXPWFP motif within SHLD3 occludes the REV1-binding surface on closed REV7, segregating shieldin from the translesion synthesis machinery, while the assembled complex recruits CST–Polα to fill in resected ssDNA (PMID:32332881, PMID:30022158). TRIP13 ATPase, facilitated by p31comet, disassembles shieldin by extracting REV7 from SHLD3, switching the repair pathway toward homologous recombination, and CHAMP1 competes with SHLD3 for REV7 binding to further regulate this balance (PMID:33597306, PMID:33051298, PMID:36044844).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2018 High

    Identification of SHLD3 as a shieldin subunit resolved how the 53BP1–RIF1 pathway physically suppresses DNA end resection: SHLD3 localizes to DSBs in a 53BP1/RIF1-dependent manner and is required for NHEJ, class-switch recombination, and PARP inhibitor sensitivity in BRCA1-deficient cells.

    Evidence Co-IP, siRNA/knockout, NHEJ reporters, CSR assays, and PARP inhibitor sensitivity across three independent labs (Nature 2018)

    PMID:30022158 PMID:30022168 PMID:30046110

    Open questions at the time
    • Structural basis of SHLD3–REV7 interaction unknown
    • Mechanism of shieldin recruitment by RIF1 not defined
    • How shieldin protects ssDNA ends mechanistically unclear
  2. 2018 High

    Discovery that shieldin recruits CST–Polα to DSBs established that end protection involves fill-in synthesis on resected ssDNA, not just physical blocking of nucleases.

    Evidence Co-IP of CST with shieldin; Polα localization at damage sites by IF; resection and RAD51 loading assays

    PMID:30022158

    Open questions at the time
    • Direct binding interface between shieldin and CST undefined
    • Whether SHLD3 itself contacts CST or acts only through SHLD1/SHLD2 unclear
  3. 2019 High

    Crystal structures of the REV7–SHLD3 RBD complex revealed the structural basis for their tight interaction: a ladle-shaped binding domain with the REV7 safety belt trapping SHLD3 and retarding dissociation, explaining the slow-off kinetics.

    Evidence 2.2–2.3 Å crystal structures; mutagenesis; binding kinetics

    PMID:31796627

    Open questions at the time
    • Structure of the full heterotetrameric shieldin complex not yet resolved
    • How the conformational state of REV7 (open vs. closed) contributes to complex assembly unknown
  4. 2020 High

    The ternary SHLD3–REV7–SHLD2 crystal structure revealed that SHLD3 nucleates a closed–open REV7 conformational heterodimer and that its FXPWFP motif occludes the REV1-binding site on REV7, explaining how shieldin is segregated from the TLS pathway.

    Evidence Crystal structure of ternary complex; competitive binding assays; NHEJ reporter assays; mutagenesis of REV7 dimerization interface

    PMID:32332881

    Open questions at the time
    • Mechanism by which TRIP13 disassembles the heterodimer not structurally characterized
    • Role of conformational heterodimer in ssDNA end protection unclear
  5. 2020 Medium

    TRIP13 ATPase was shown to disassemble shieldin by dissociating REV7 from SHLD3, with p31comet facilitating the TRIP13–REV7 interaction, establishing the first known disassembly mechanism for the complex and explaining how HR can be re-enabled.

    Evidence Co-IP; chromatin fractionation; HR/NHEJ reporters; PARP inhibitor resistance assays

    PMID:33051298

    Open questions at the time
    • Structural mechanism of TRIP13-mediated extraction not yet visualized
    • Whether TRIP13 regulation is cell-cycle-dependent not tested
  6. 2021 High

    Cryo-EM of the TRIP13–SHLD2–SHLD3–REV7 complex revealed the structural mechanism of disassembly: TRIP13 threads the N-terminus of closed REV7 through its central pore using ATP hydrolysis-driven rotatory translocation, directly visualizing how the shieldin complex is dismantled.

    Evidence Crystal structures and cryo-EM of ATPγS-bound TRIP13–shieldin complexes; mutagenesis; biochemical disassembly assays

    PMID:33597306

    Open questions at the time
    • Regulation of TRIP13 activity at DSBs in vivo not characterized
    • Whether disassembly is processive or stochastic in cells unknown
  7. 2021 High

    REV7 dimerization mediated by SHLD2 was shown to accelerate its association with SHLD3, indicating that complex assembly is cooperative rather than strictly sequential and linking dimerization to functional NHEJ output.

    Evidence Biochemical assembly assays; NHEJ reporters; dimerization-defective REV7 mutants

    PMID:34521823

    Open questions at the time
    • Kinetic rate constants for the cooperative step not determined
    • Whether post-translational modifications regulate cooperativity unknown
  8. 2022 Medium

    CHAMP1 was identified as a competitor of SHLD3 for REV7 seatbelt binding, establishing REV7 as a binary molecular switch: SHLD3 binding promotes NHEJ, while CHAMP1 binding promotes HR by displacing shieldin.

    Evidence Direct binding assays; Co-IP; HR/NHEJ reporters; end-resection assays

    PMID:36044844

    Open questions at the time
    • Not independently replicated; single lab study
    • Whether CHAMP1–REV7 interaction is regulated by cell cycle or DNA damage signaling unknown
    • Structural basis of CHAMP1 competition with SHLD3 not determined
  9. 2023 High

    The direct RIF1–SHLD3 interaction was mapped to the RIF1 HEAT-repeat domain and the SHLD3 eIF4E-like domain, resolving how shieldin is recruited to 53BP1-marked DSBs upstream of REV7 engagement.

    Evidence AlphaFold2-Multimer prediction validated by in vitro pulldown; DSB localization by IF; CSR and PARP inhibitor sensitivity assays

    PMID:37306046

    Open questions at the time
    • Experimental atomic-resolution structure of RIF1–SHLD3 interface not yet available
    • Whether phosphorylation of RIF1 regulates SHLD3 binding untested
  10. 2023 Medium

    Kinetic reconstitution showed that the first REV7 binding to SHLD3 is remarkably slow and rate-limiting, while the second REV7 (with SHLD2) binds rapidly without structural remodeling, and SHLD3 harbors an N-terminal DNA-binding domain, revealing a two-step assembly mechanism with potential direct DNA engagement.

    Evidence Fluorescence-based binding kinetics; DNA binding assays; in vitro reconstitution

    PMID:37031298

    Open questions at the time
    • Functional relevance of SHLD3 DNA binding in vivo untested
    • Whether DNA binding contributes to DSB localization independent of RIF1 unknown
  11. 2025 Medium

    Genetic epistasis between CTC1 and SHLD1 during CSR demonstrated that shieldin and CST function in the same pathway to prevent excessive end resection, but the specific SHLD1-CTC1 physical interaction via the LDLP motif is dispensable for CSR, suggesting redundant recruitment mechanisms.

    Evidence Double knockout B cell lines; CSR assays; SHLD1 separation-of-function mutant complementation

    PMID:40178294

    Open questions at the time
    • Alternative CST recruitment mechanism to shieldin not identified
    • Whether SHLD3 directly participates in CST recruitment remains unresolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the functional significance of SHLD3's intrinsic DNA-binding activity in vivo, how post-translational modifications and cell-cycle signals regulate shieldin assembly and disassembly dynamics at DSBs, and whether an experimental high-resolution structure of the full RIF1–SHLD3 interface can be obtained.
  • No in vivo validation of SHLD3 DNA-binding function
  • Cell-cycle regulation of SHLD3–REV7 binding kinetics not characterized
  • Full heterotetramer structure in complex with RIF1 not resolved experimentally

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 4 GO:0003677 DNA binding 1
Localization
GO:0005694 chromosome 2
Pathway
R-HSA-73894 DNA Repair 6 R-HSA-168256 Immune System 3
Partners
CHAMP1REV7RIF1SHLD1SHLD2TRIP13
Complex memberships
Shieldin (SHLD3-REV7-SHLD2-SHLD1)

Evidence

Reading pass · 12 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2018 SHLD3 (CTC-534A2.2/FLJ26957) is a subunit of the shieldin complex (REV7-SHLD1-SHLD2-SHLD3) that localizes to DNA double-strand break sites in a 53BP1- and RIF1-dependent manner, where it promotes non-homologous end joining and suppresses DNA end resection. Co-immunoprecipitation, loss-of-function (siRNA/knockout) with phenotypic readouts (NHEJ efficiency, resection assays, immunoglobulin class-switch recombination, PARP inhibitor sensitivity) Nature High 30022158 30022168 30046110
2018 SHLD3 recruits REV7 to DSB sites as the apical subunit of shieldin; the interaction is mediated by a REV7-binding domain (RBD) on SHLD3 containing an N-terminal loop and a C-terminal αC-helix, both indispensable for high-affinity (low-nanomolar) REV7 binding, with the REV7 'safety belt' region retarding dissociation. Crystal structures of REV7–SHLD3 RBD complex at 2.2–2.3 Å; in vitro and in vivo binding assays; binding kinetics analyses The Journal of biological chemistry High 31796627
2020 Crystal structure of the SHLD3-REV7-SHLD2 ternary complex revealed that SHLD3 drives formation of an unexpected conformational C(closed)-REV7–O(open)-REV7 homodimer; SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 via β-sheet sandwich. The conserved FXPWFP motif of SHLD3 binds C-REV7 and blocks REV1 binding, thereby excluding shieldin from the REV1/Pol ζ TLS complex. Crystal structure; mutagenesis disrupting REV7 conformational dimer; NHEJ efficiency assays; competitive binding assays Nature communications High 32332881
2021 Crystal and cryo-EM structures of SHLD3-REV7 and SHLD2-SHLD3-REV7 complexes confirmed that shieldin assembly requires SHLD2-SHLD3-induced conformational heterodimerization of open and closed REV7 forms; TRIP13 ATPase disassembles shieldin by inserting the N-terminus of C-REV7 into its central channel and pulling the unfolded peptide through via ATP hydrolysis-driven rotatory motions. Crystal structures; cryo-EM of ATPγS-bound SHLD2-SHLD3-REV7-TRIP13 complexes; mutagenesis; biochemical 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; dimerization-defective MAD2L2 impairs shieldin assembly and NHEJ. SHLD3 presence, together with MAD2L2 dimerization, allows shieldin to interact with TRIP13 ATPase, which controls shieldin (dis)assembly and activity in DNA repair. Biochemical assembly assays; NHEJ reporter assays; co-immunoprecipitation; dimerization-defective MAD2L2 mutants; TRIP13 interaction assays Nature communications High 34521823
2018 The 53BP1-RIF1-shieldin (REV7-SHLD1-SHLD2-SHLD3) pathway recruits CST (CTC1-STN1-TEN1) and Polα to DSB sites in a shieldin-dependent manner; CST-Polα-mediated fill-in of resected ssDNA helps limit end resection and suppresses homologous recombination, with CST interacting directly with shieldin. Co-immunoprecipitation of CST with shieldin; localization of Polα to damage sites by immunofluorescence; depletion studies measuring resection; RAD51 loading assays; PARP inhibitor sensitivity in BRCA1-deficient cells Nature High 30022158
2020 TRIP13 ATPase remodels REV7, causing its dissociation from the shieldin subunit SHLD3, thereby inactivating shieldin and promoting DNA end resection and homologous recombination; p31comet facilitates this by promoting the TRIP13-REV7 interaction and participates in extraction of REV7 from chromatin. Co-immunoprecipitation; chromatin fractionation; HR/NHEJ reporter assays; PARP inhibitor resistance assays Proceedings of the National Academy of Sciences of the United States of America Medium 33051298
2023 AlphaFold2-Multimer predicted, and in vitro pulldown and cellular assays confirmed, a direct interaction between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3; this RIF1-SHLD3 binding is essential for shieldin recruitment to DSB sites, antibody class switch recombination, and PARP inhibitor sensitivity. AlphaFold2-Multimer structural prediction; in vitro pulldown assays; cellular localization assays (immunofluorescence at DSBs); class switch recombination assays; PARP inhibitor sensitivity assays EMBO reports High 37306046
2022 CHAMP1 competes with SHLD3 for binding to the REV7 seatbelt domain; CHAMP1 binding to REV7 reduces shieldin complex levels and promotes DSB end resection and homologous recombination, establishing REV7 as a molecular switch between NHEJ (via SHLD3) and HR (via CHAMP1). Direct binding assays; Co-immunoprecipitation; end-resection assays; HR/NHEJ reporter assays Cell reports Medium 36044844
2023 SHLD3 harbors a promiscuous DNA-binding domain at its N-terminus; the interaction of SHLD3 with the first REV7 molecule is remarkably slow (rate-limiting step), while association with a second REV7 (together with SHLD2) is fast and does not require structural remodeling, providing insight into the kinetic mechanism of shieldin assembly. Biochemical binding kinetics (fluorescence-based assays); DNA binding assays; in vitro reconstitution of shieldin assembly Communications biology Medium 37031298
2021 Cryo-EM structure of apo Pol ζ combined with crystal structure of human REV7 bound to a fragment of SHLD3 protein revealed the molecular interactions by which REV7 recognizes partner proteins including SHLD3 in the context of shieldin. Cryo-EM (apo Pol ζ); crystal structure of REV7-SHLD3 fragment The Journal of biological chemistry Medium 34174285
2025 CTC1 (CST) and SHLD1 are epistatic in preventing exacerbated DNA end resection during class switch recombination; a SHLD1 mutant defective in CST binding (SHLD1ΔLDLP) is fully proficient for class switching, demonstrating that the SHLD1-CTC1 interaction through this specific motif is dispensable for CST and shieldin functions in promoting CSR. Genetic epistasis (double knockout B cell lines); CSR assays; end-resection analysis; complementation with SHLD1 separation-of-function 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 500 30022168
2018 53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in. Nature 350 30022158
2018 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ. Nature 252 30046110
2019 Shieldin - the protector of DNA ends. EMBO reports 186 30948458
2020 Molecular basis for assembly of the shieldin complex and its implications for NHEJ. Nature communications 42 32332881
2021 MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair. Nature communications 33 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