Affinage

RADX

RPA-related protein RADX · UniProt Q6NSI4

Length
855 aa
Mass
97.6 kDa
Annotated
2026-04-28
11 papers in source corpus 9 papers cited in narrative 9 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RADX is an OB-fold-containing ssDNA-binding protein that functions as a homo-oligomeric (predominantly trimeric) regulator of RAD51 at replication forks, balancing fork protection against excessive fork reversal and collapse (PMID:28735897, PMID:38466836). RADX antagonizes RAD51 through multiple coordinated mechanisms: it competes with RAD51 for ssDNA occupancy, condenses RPA-coated ssDNA to block RAD51 loading, directly binds ATP-bound RAD51 to stimulate its ATPase activity and destabilize nucleofilaments, and caps the ends of RAD51 filaments (PMID:30021152, PMID:32621611, PMID:33453169, PMID:38466836). This antagonism is counteracted by BRCA2, establishing a regulatory axis in which RADX confines RAD51-dependent fork reversal to persistently stalled forks while preventing RAD51-driven replication slowing at elongating forks; accordingly, RADX depletion restores fork protection in BRCA1-, BRCA2-, FANCA-, FANCD2-, or BOD1L-deficient cells (PMID:30021152, PMID:34107305). At persistently stalled forks, RADX removes inhibitory RAD51 to facilitate SMARCAL1-dependent fork reversal, and separation-of-function mutants that retain DNA and RAD51 binding but lose the ability to stimulate RAD51 ATP hydrolysis cause RAD51 chromatin accumulation and replication defects, demonstrating that RAD51 turnover is a critical effector output (PMID:34107305, PMID:37572935).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 2017 High

    The identification of RADX as a replication-fork-associated ssDNA-binding protein that opposes RAD51 established the existence of a dedicated antagonist restraining RAD51 activity during normal replication.

    Evidence iPOND proteomics, siRNA knockdown, DNA fiber assays, and DSB detection in human cells

    PMID:28735897 PMID:29021206

    Open questions at the time
    • Mechanism by which RADX antagonizes RAD51 (competition vs. direct interaction) was unresolved
    • Whether RADX acts alone or in complex was unknown
    • Structural basis of OB-fold-mediated ssDNA binding was not determined
  2. 2018 High

    Genetic epistasis experiments demonstrated that RADX and RAD51 compete on ssDNA and that RADX loss restores fork protection in multiple HR/FA-deficient backgrounds, positioning RADX as a tunable counterweight to BRCA2-dependent RAD51 loading.

    Evidence siRNA epistasis with BRCA1, BRCA2, FANCA, FANCD2, BOD1L; in vitro ssDNA competition binding; nuclease epistasis

    PMID:30021152

    Open questions at the time
    • Whether RADX physically contacts RAD51 or only competes for ssDNA was untested
    • In vivo relevance of competition at physiological protein concentrations was unclear
  3. 2020 High

    Single-molecule imaging revealed that RADX forms higher-order assemblies that condense ssDNA and block RPA displacement by RAD51, providing a biophysical mechanism for how RADX prevents RAD51 nucleation even on RPA-coated substrates.

    Evidence Single-molecule ssDNA curtains with TIRF microscopy using purified proteins at physiological ratios

    PMID:32621611

    Open questions at the time
    • Whether RADX directly contacts RAD51 protein in addition to competing for ssDNA was still unknown
    • Oligomeric state and stoichiometry of RADX assemblies were not resolved
  4. 2021 High

    Biochemical reconstitution showed that RADX directly binds ATP-bound RAD51, stimulates RAD51 ATPase activity to destabilize filaments, and inhibits strand exchange and D-loop formation — revealing an active enzymatic disruption mechanism beyond simple ssDNA competition — while BRCA2 can overcome this inhibition.

    Evidence In vitro strand exchange, D-loop, and ATPase assays with purified proteins; RAD51 active-site mutagenesis; pulldown

    PMID:33453169

    Open questions at the time
    • Structural basis of the RADX–RAD51 interaction was unknown
    • Which RADX domains mediate RAD51 binding was not mapped
  5. 2021 High

    Demonstrating context-dependent function, RADX was shown to promote SMARCAL1-dependent fork reversal at persistently stalled forks by removing inhibitory RAD51, while restraining fork reversal at elongating forks — resolving the apparent paradox of how one factor both inhibits and facilitates fork remodeling.

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

    PMID:34107305

    Open questions at the time
    • How RADX distinguishes persistently stalled from transiently stalled forks was not explained
    • Signaling inputs that regulate RADX recruitment kinetics remain undefined
  6. 2022 High

    Structure–function analysis established that RADX oligomerization via C-terminal surfaces is essential for its replication fork regulatory activity, as oligomerization-defective mutants fail to control fork stability but are rescued by a heterologous dimerization domain.

    Evidence Biochemical oligomerization assays, C-terminal mutagenesis, heterologous dimerization domain complementation, DNA fiber assays

    PMID:35120927

    Open questions at the time
    • Atomic-resolution structure of the oligomer was not available
    • Whether oligomerization is required specifically for RAD51 interaction or ssDNA condensation was not dissected
  7. 2023 High

    CRISPR base-editing screens isolated separation-of-function mutants proving that stimulation of RAD51 ATP hydrolysis — not merely ssDNA binding or RAD51 interaction — is the critical effector activity through which RADX maintains genome stability during replication.

    Evidence CRISPR base-editing tiling mutagenesis, RAD51 chromatin fractionation, ATPase assays, DNA damage sensitivity assays in human cells

    PMID:37572935

    Open questions at the time
    • Residues in RADX that contact RAD51's ATPase domain were not mapped
    • Whether these mutants alter fork reversal specifically was not tested
  8. 2024 High

    The cryo-EM structure of RADX at near-atomic resolution revealed the molecular basis of multivalent ssDNA binding and concentration-dependent oligomerization, and negative-stain EM visualized RADX oligomers capping the ends of RAD51 filaments, providing a structural explanation for filament destabilization.

    Evidence Cryo-EM structure determination (2–4 Å), mass photometry, negative stain EM of RADX–RAD51 filament complexes

    PMID:38466836

    Open questions at the time
    • High-resolution structure of the RADX–RAD51 interface is not yet available
    • How RADX capping leads to filament disassembly mechanistically (directional stripping vs. end-dependent destabilization) is unresolved
    • Post-translational regulation of RADX oligomerization in vivo has not been characterized

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key open questions include the structural basis of the RADX–RAD51 direct interaction, the signaling mechanisms that regulate RADX recruitment to distinguish elongating from persistently stalled forks, and whether RADX dysfunction contributes to human disease.
  • No high-resolution structure of the RADX–RAD51 complex exists
  • Post-translational modifications regulating RADX activity in vivo are unknown
  • No disease association has been established through patient studies

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003677 DNA binding 4 GO:0098772 molecular function regulator activity 3
Localization
GO:0005654 nucleoplasm 2 GO:0005694 chromosome 2
Pathway
R-HSA-69306 DNA Replication 4 R-HSA-73894 DNA Repair 3
Partners
BRCA2RAD51RPARTEL1SMARCAL1

Evidence

Reading pass · 9 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2017 RADX is an RPA-like, single-strand DNA binding protein (with OB fold domains) that is recruited to replication forks, where it antagonizes RAD51 to prevent excessive fork reversal and fork collapse; inactivation of RADX leads to excessive RAD51 activity, slowed replication elongation, and double-strand breaks. iPOND (isolation of proteins on nascent DNA), siRNA knockdown, DNA fiber assays, DSB detection Molecular cell High 28735897
2017 RADX binds ssDNA via an N-terminal OB fold cluster and is recruited to sites of replication stress; deregulation of RADX ssDNA binding causes enhanced replication fork stalling and degradation; a balanced interplay between RADX and RPA ssDNA-binding activities is critical for replication integrity. ssDNA pulldown, OB fold mutagenesis, siRNA knockdown, DNA fiber assays, immunofluorescence EMBO reports High 29021206
2018 RADX antagonizes RAD51 to modulate stalled fork protection; silencing RADX restores fork protection in BRCA1-, BRCA2-, FANCA-, FANCD2-, or BOD1L-deficient cells; RADX overexpression causes MRE11- and DNA2-dependent fork degradation; RADX competes with RAD51 for binding to ssDNA. siRNA knockdown, DNA fiber assays, ssDNA competition binding assay, epistasis with nuclease mutants Cell reports High 30021152
2020 RADX condenses ssDNA filaments via higher-order assemblies that can capture ssDNA in trans, even when ssDNA is coated with RPA at physiological ratios; RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Single-molecule fluorescence imaging in vitro, ssDNA curtains assay, TIRF microscopy Nucleic acids research High 32621611
2021 RADX directly interacts with ATP-bound RAD51, stimulates RAD51 ATP hydrolysis, and destabilizes RAD51 nucleofilaments, thereby inhibiting RAD51 strand exchange and D-loop formation; BRCA2 can overcome RADX-dependent RAD51 inhibition; RADX ssDNA binding and RAD51 interaction are both required for maintaining fork elongation rates and stability. In vitro strand exchange assay, D-loop assay, ATPase assay, active-site mutagenesis, DNA fiber assay, pulldown Molecular cell High 33453169
2021 RADX context-dependently inhibits fork reversal at elongating forks (to prevent unnecessary slowing) but promotes fork reversal at persistently stalled forks; RADX directly enhances SMARCAL1-dependent fork reversal when pre-bound RAD51 on model fork substrates is inhibitory; inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1. DNA fiber assay, electron microscopy of replication intermediates, in vitro fork reversal reconstitution with SMARCAL1, epistasis with RTEL1 KO Molecular cell High 34107305
2022 RADX functions as a homo-oligomer; it uses at least two oligomerization surfaces including a C-terminal region; mutations in this region prevent oligomerization and abolish RADX's ability to regulate replication fork stability in cells; addition of a heterologous dimerization domain to oligomerization mutants restores function. Biochemical oligomerization assay, structure-function mutagenesis, complementation with heterologous dimerization domain, DNA fiber assay The Journal of biological chemistry High 35120927
2023 CRISPR base-editing screens identified separation-of-function RADX mutants that bind DNA and RAD51 but have reduced ability to stimulate RAD51 ATP hydrolysis; cells expressing these mutants accumulate RAD51 on chromatin and exhibit replication defects, indicating that RADX must promote RAD51 ATP turnover to regulate genome stability during replication. CRISPR base-editing tiling screen, RAD51 chromatin fractionation, ATPase assays, DNA damage sensitivity assays Journal of molecular biology High 37572935
2024 Cryo-EM structure of RADX determined ab initio reveals the molecular basis for RADX oligomerization and multi-valent ssDNA binding; RADX forms concentration-dependent oligomeric states with predominant trimers in the presence of ssDNA; negative stain EM shows RADX oligomers positioned at the ends of RAD51 filaments, supporting a filament-capping mechanism of RAD51 inhibition. Cryo-EM structure determination (2–4 Å), mass photometry, negative stain EM of RADX–RAD51 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 163 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 31 33453169
2017 RADX interacts with single-stranded DNA to promote replication fork stability. EMBO reports 29 29021206
2021 RADX prevents genome instability by confining replication fork reversal to stalled forks. Molecular cell 27 34107305
2020 RADX condenses single-stranded DNA to antagonize RAD51 loading. Nucleic acids research 19 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 5 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 2 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