Affinage

RADX

RPA-related protein RADX · UniProt Q6NSI4

Length
855 aa
Mass
97.6 kDa
Annotated
2026-06-10
11 papers in source corpus 9 papers cited in narrative 9 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RADX is an RPA-like single-stranded DNA (ssDNA)-binding protein that operates at replication forks to restrain RAD51 activity and thereby preserve genome stability (PMID:28735897). It binds ssDNA through an N-terminal OB-fold cluster that mediates its recruitment to sites of replication stress, and its activity is balanced against that of RPA to maintain replication integrity (PMID:29021206). RADX antagonizes RAD51 by two cooperating mechanisms: it competes with RAD51 for ssDNA and condenses RPA-coated ssDNA into higher-order assemblies that block RPA displacement and prevent RAD51 loading (PMID:30021152, PMID:32621611), and it directly engages ATP-bound RAD51 to stimulate RAD51 ATP hydrolysis, destabilize RAD51 nucleofilaments, and inhibit strand exchange and D-loop formation — an activity that BRCA2 can override, establishing RADX and BRCA2 as opposing regulators of filament stability (PMID:33453169, PMID:37572935). Through this regulation RADX confines fork reversal: it suppresses reversal at elongating forks to prevent collapse but promotes SMARCAL1-dependent reversal at persistently stalled forks when pre-bound RAD51 is inhibitory (PMID:34107305). RADX functions as a homo-oligomer — predominantly a trimer on ssDNA — assembled via multiple interaction surfaces including a C-terminal region, and structural analysis indicates it caps and restricts RAD51 filament ends (PMID:35120927, PMID:38466836).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2017 High

    Established RADX as a previously uncharacterized fork-associated ssDNA-binding factor whose loss destabilizes replication, framing it as a RAD51 antagonist rather than a generic fork protein.

    Evidence Proteomic identification at forks with siRNA/CRISPR depletion, DNA fiber assays, and γH2AX foci

    PMID:28735897

    Open questions at the time
    • Did not resolve whether antagonism is via ssDNA competition or direct RAD51 binding
    • No structural or biochemical mechanism for fork recruitment
  2. 2017 High

    Mapped the ssDNA-binding determinant to an N-terminal OB-fold cluster and tied that binding to stress-site recruitment, defining the biochemical basis of RADX localization.

    Evidence In vitro ssDNA binding with OB-fold deletion/mutation, immunofluorescence, and DNA fiber assays

    PMID:29021206

    Open questions at the time
    • Did not establish how RADX-RPA balance is set quantitatively
    • Did not test direct RAD51 contact
  3. 2018 High

    Showed RADX competes with RAD51 for ssDNA and that RAD51 dosage dictates fork fate, explaining why RADX loss rescues fork protection in BRCA/Fanconi-deficient cells.

    Evidence DNA fiber assays across multiple BRCA/Fanconi-deficient lines, overexpression, and nuclease-inhibitor experiments

    PMID:30021152

    Open questions at the time
    • Competition demonstrated functionally but not at single-molecule resolution
    • Direct RADX-RAD51 protein interaction not yet shown
  4. 2020 High

    Resolved the ssDNA-level mechanism by showing RADX condenses even RPA-coated ssDNA and blocks RPA displacement, recasting RADX as an ssDNA condensation protein that prevents RAD51 loading.

    Evidence Single-molecule TIRF imaging and ssDNA curtains with purified proteins

    PMID:32621611

    Open questions at the time
    • Did not address whether RADX also acts on assembled RAD51 filaments
    • In vitro ratios may not capture in vivo regulation
  5. 2021 High

    Identified a second, direct mechanism: RADX binds ATP-bound RAD51, stimulates its ATPase, destabilizes nucleofilaments and blocks strand exchange, and placed RADX opposite BRCA2 in RAD51 filament regulation.

    Evidence In vitro strand exchange, D-loop and ATPase assays, ATP-state-selective interaction studies, and BRCA2 epistasis with fiber assays

    PMID:33453169

    Open questions at the time
    • Structural basis of the ATP-bound-RAD51 interaction not defined
    • Quantitative interplay with the ssDNA-condensation activity unresolved
  6. 2021 High

    Reconciled RADX's apparently opposing roles by showing it inhibits reversal at elongating forks but promotes SMARCAL1-dependent reversal at persistently stalled forks, making fork outcome stress-dependent.

    Evidence DNA fiber assays, electron microscopy of replication intermediates, RTEL1 epistasis, and in vitro SMARCAL1 fork reversal reconstitution

    PMID:34107305

    Open questions at the time
    • Signal that switches RADX between inhibitory and promoting modes is unknown
    • Threshold defining 'persistent' stalling not defined
  7. 2022 High

    Demonstrated that homo-oligomerization, via multiple surfaces including a C-terminal region, is obligatory for RADX function in cells.

    Evidence Oligomerization mapping with mutagenesis, heterologous dimerization-domain rescue, and DNA fiber assays

    PMID:35120927

    Open questions at the time
    • Oligomeric stoichiometry on ssDNA not yet defined
    • Did not resolve oligomer architecture structurally
  8. 2023 High

    Used separation-of-function mutants to prove that RADX must actively promote RAD51 ATP turnover — not merely bind DNA and RAD51 — to maintain genome stability.

    Evidence CRISPR base-editing screen with ATPase, chromatin fractionation, fiber, and damage-sensitivity assays

    PMID:37572935

    Open questions at the time
    • Atomic mechanism by which RADX stimulates RAD51 ATPase not defined
    • Did not separate ssDNA-condensation from ATPase-stimulation contributions in vivo
  9. 2024 High

    Provided the first RADX structure, defining oligomerization and multivalent ssDNA binding and visualizing RADX capping RAD51 filament ends, giving a physical model for filament restriction.

    Evidence Ab initio cryo-EM at 2–4 Å, mass photometry, and negative-stain EM of RADX-RAD51 filament complexes

    PMID:38466836

    Open questions at the time
    • No high-resolution structure of the RADX-RAD51 interface
    • Capping model not yet tested with structure-guided interface mutants in cells

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the two RADX activities (ssDNA condensation versus direct RAD51 ATPase stimulation/filament capping) are coordinated and toggled by replication-stress signals to choose fork elongation versus reversal remains unresolved.
  • No mechanism for sensing 'persistent' stalling
  • No structure of the RADX-RAD51 complex interface
  • Upstream regulation/post-translational control of RADX unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003677 DNA binding 5 GO:0098772 molecular function regulator activity 4 GO:0140313 molecular sequestering activity 2
Pathway
R-HSA-69306 DNA Replication 4 R-HSA-73894 DNA Repair 3
Partners
BRCA2RAD51RPASMARCAL1

Evidence

Reading pass · 9 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2017 RADX was identified as an RPA-like, single-strand DNA (ssDNA) binding protein that is recruited to replication forks, where it antagonizes RAD51 to prevent fork collapse. Inactivation of RADX causes excessive RAD51 activity, leading to slowed replication elongation and double-strand breaks. Proteomic identification at replication forks, genetic depletion (siRNA/CRISPR), DNA fiber assays, γH2AX foci Molecular cell High 28735897
2017 RADX binds ssDNA via an N-terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Disruption of RADX expression or ssDNA binding leads to enhanced replication fork stalling and degradation. A balanced interplay between RADX and RPA ssDNA-binding activities is critical for replication integrity. Biochemical ssDNA binding assays, OB-fold deletion/mutation analysis, immunofluorescence at replication stress sites, DNA fiber assays EMBO reports High 29021206
2018 RADX antagonizes RAD51 at stalled replication forks by competing with RAD51 for binding to ssDNA. Silencing RADX restores fork protection in cells deficient for BRCA1, BRCA2, FANCA, FANCD2, or BOD1L. RADX overexpression causes fork degradation dependent on MRE11 and DNA2 nucleases and requires fork reversal. The level of RAD51 determines fork fate, with more RAD51 required for protection than reversal. DNA fiber assays, siRNA knockdown in multiple BRCA/Fanconi pathway-deficient cell lines, RADX overexpression, nuclease inhibitor experiments Cell reports High 30021152
2020 RADX condenses ssDNA filaments even when coated with RPA at physiological protein ratios, forming higher-order assemblies that can capture ssDNA in trans. RADX blocks RPA displacement by RAD51 and prevents RAD51 loading onto ssDNA, acting as an ssDNA condensation protein. Single-molecule imaging (total internal reflection fluorescence microscopy), in vitro reconstitution with purified proteins, single-molecule curtains Nucleic acids research High 32621611
2021 RADX directly inhibits RAD51 strand exchange and D-loop formation. RADX selectively interacts with ATP-bound RAD51, stimulates RAD51 ATP hydrolysis, and destabilizes RAD51 nucleofilaments. Both ssDNA binding and direct RAD51 interaction are required for RADX to maintain replication fork elongation rates. BRCA2 can overcome RADX-dependent RAD51 inhibition, establishing RADX and BRCA2 as opposing regulators of RAD51 nucleofilament stability. In vitro strand exchange assay, D-loop assay, ATPase assay, biochemical interaction studies with ATP-bound RAD51, genetic complementation with BRCA2, DNA fiber assays Molecular cell High 33453169
2021 RADX can either inhibit or promote fork reversal depending on replication stress levels: it inhibits fork reversal at elongating forks to prevent collapse, but at persistently stalled forks, RADX localizes to promote reversed fork structure formation. RADX increases SMARCAL1-dependent fork reversal when pre-bound RAD51 is inhibitory, acting via direct interaction with both RAD51 and ssDNA. DNA fiber assays, electron microscopy of replication intermediates, genetic epistasis with RTEL1 and fork protection factors, in vitro fork reversal assay with SMARCAL1 and RADX Molecular cell High 34107305
2022 RADX functions as a homo-oligomer to regulate replication fork stability. RADX oligomerizes via at least two interaction surfaces, including a C-terminal region. Mutations preventing oligomerization abolish RADX function in cells, and this can be rescued by addition of a heterologous dimerization domain. Biochemical oligomerization assays, mutagenesis of oligomerization surfaces, complementation with heterologous dimerization domain, DNA fiber assays in cells The Journal of biological chemistry High 35120927
2023 CRISPR base editing screen identified RADX separation-of-function mutants that bind DNA and RAD51 but have reduced ability to stimulate RAD51 ATP hydrolysis. Cells expressing these mutants accumulate RAD51 on chromatin, exhibit replication defects, accumulate DNA damage, and are hypersensitive to replication stress, indicating that RADX must promote RAD51 ATP turnover to regulate genome stability during DNA replication. CRISPR base editing screen, ATPase assay, chromatin fractionation, DNA fiber assay, DNA damage sensitivity assays Journal of molecular biology High 37572935
2024 Cryo-EM structure of RADX (no structurally characterized orthologs) determined ab initio at 2–4 Å resolution. RADX forms concentration-dependent oligomeric states (predominantly trimers in the presence of ssDNA). The structure reveals the molecular basis for oligomerization and multivalent ssDNA binding. Negative stain EM imaging shows a RADX oligomer at the end of RAD51 filaments, supporting a model in which RADX caps and restricts RAD51 filament ends. Cryo-EM structure determination, mass photometry (oligomeric state analysis), negative stain EM of RADX-RAD51 filament complexes Proceedings of the National Academy of Sciences of the United States of America High 38466836

Source papers

Stage 0 corpus · 11 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2017 RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks. Molecular cell 165 28735897
2018 RADX Modulates RAD51 Activity to Control Replication Fork Protection. Cell reports 84 30021152
2021 RADX controls RAD51 filament dynamics to regulate replication fork stability. Molecular cell 32 33453169
2021 RADX prevents genome instability by confining replication fork reversal to stalled forks. Molecular cell 29 34107305
2017 RADX interacts with single-stranded DNA to promote replication fork stability. EMBO reports 29 29021206
2020 RADX condenses single-stranded DNA to antagonize RAD51 loading. Nucleic acids research 20 32621611
2024 Structure of RADX and mechanism for regulation of RAD51 nucleofilaments. Proceedings of the National Academy of Sciences of the United States of America 6 38466836
2022 Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability. The Journal of biological chemistry 5 35120927
2023 CRISPR-dependent Base Editing Screens Identify Separation of Function Mutants of RADX with Altered RAD51 Regulatory Activity. Journal of molecular biology 3 37572935
2023 Structure of RADX and mechanism for regulation of RAD51 nucleofilaments. bioRxiv : the preprint server for biology 1 37786681
2023 RADX Gene Variant May Predispose to Familial Asperger Syndrome. Genes 0 36833228

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