Affinage

RNF169

E3 ubiquitin-protein ligase RNF169 · UniProt Q8NCN4

Length
708 aa
Mass
77.2 kDa
Annotated
2026-06-10
9 papers in source corpus 8 papers cited in narrative 7 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

RNF169 is a chromatin-associated factor that biases DNA double-strand break (DSB) repair toward resection-dependent pathways by reading RNF168-deposited ubiquitin marks (PMID:22492721, PMID:22733822). It accumulates at DSB foci by recognizing RNF168-catalyzed ubiquitylation of H2A-Lys13/Lys15, engaging the modified nucleosome through a three-pronged interaction: a canonical ubiquitin-binding helix plus two arginine-rich motifs that contact the nucleosome acidic patch (PMID:22492721, PMID:22733822, PMID:28406400). Once recruited, RNF169 competitively displaces 53BP1 and RAP80-BRCA1 from RNF168-modified chromatin independently of its own E3 ligase catalytic activity, alleviating the 53BP1 barrier to end resection and thereby promoting CtIP-dependent resection, homologous recombination, and single-strand annealing over non-homologous end joining (PMID:22492721, PMID:22733822, PMID:30104380). Its nuclear import and protein stability are coupled through a dual-function nuclear localization signal that also binds the deubiquitylase USP7; disrupting this interaction destabilizes RNF169, impairs high-fidelity DSB repair, and sensitizes cells to PARP inhibition (PMID:28325877). RNF169's 53BP1-displacing activity is further tuned by DYRK1A-mediated phosphorylation, which enhances 53BP1 displacement and homologous recombination (PMID:30773093, PMID:30979931). Beyond DSB repair, RNF169 acts as a reader of the H2BK120ub mark at stalled replication forks, where it protects nascent DNA from excessive nucleolytic degradation (PMID:41145912).

Mechanistic history

Synthesis pass · year-by-year structured walk · 7 steps
  1. 2012 High

    Established that RNF169 is a damage-responsive chromatin factor recruited to DSBs through ubiquitin recognition, defining its place downstream of the RNF8/RNF168 signaling cascade.

    Evidence Cellular localization, domain mapping, and loss-of-function in human cells

    PMID:22492721 PMID:22733822

    Open questions at the time
    • The structural basis of ubiquitin recognition was not resolved
    • Did not establish the functional consequence of recruitment
  2. 2012 High

    Showed that RNF169's role is to competitively restrict 53BP1 and RAP80-BRCA1 loading and shift repair toward HR, and that this is independent of catalytic E3 activity, revealing a non-enzymatic competitive mechanism.

    Evidence Overexpression/knockdown, foci competition assays, catalytic-dead mutants, and survival assays in human cells

    PMID:22492721 PMID:22733822

    Open questions at the time
    • The molecular determinants of competitive binding versus 53BP1 were not defined
    • Did not address how repair-pathway choice is quantitatively tuned
  3. 2017 High

    Resolved at atomic resolution how RNF169 reads ubiquitylated H2A-K13/K15, defining a three-pronged interaction that explains its high-affinity, mark-specific nucleosome engagement.

    Evidence Methyl-TROSY NMR, molecular dynamics, cryo-EM, and mutagenesis

    PMID:28406400

    Open questions at the time
    • Did not connect structural binding mode to in vivo 53BP1 competition kinetics
    • Did not address selectivity against other ubiquitin readers at the same mark
  4. 2017 High

    Identified a dual-function NLS that couples nuclear import to USP7 binding and protein stabilization, linking RNF169 abundance to DSB repair fidelity and PARP-inhibitor sensitivity.

    Evidence Crystal structure of USP7-RNF169 NLS peptide, structure-guided uncoupling mutagenesis, and cellular stability/repair assays

    PMID:28325877

    Open questions at the time
    • Whether USP7 directly deubiquitylates RNF169 was not established
    • Did not define how RNF169 abundance is dynamically regulated during the cell cycle
  5. 2018 High

    Demonstrated that RNF169 acts at resected, RPA-bound DSB ends to promote CtIP-dependent resection and single-strand annealing, placing it downstream of break processing rather than only at initial recruitment.

    Evidence ChIP at AsiSI-induced DSBs, SSA reporter assays, and epistasis with CtIP and 53BP1

    PMID:30104380

    Open questions at the time
    • Did not resolve how preferential accumulation at resected ends is achieved
    • The balance between HR and SSA outcomes was not mechanistically dissected
  6. 2019 Medium

    Showed that DYRK1A phosphorylates RNF169 to enhance its 53BP1-displacing activity, identifying a post-translational input that tunes repair-pathway choice.

    Evidence AP-MS, in vitro kinase assay, phosphosite mutagenesis, and 53BP1 foci quantification in U-2 OS cells

    PMID:30773093 PMID:30979931

    Open questions at the time
    • Functional interpretation of individual phosphosites differs between studies
    • Upstream signals activating DYRK1A toward RNF169 are unknown
  7. 2025 Medium

    Extended RNF169 function beyond DSBs by identifying it as an H2BK120ub reader at stalled replication forks required to protect nascent DNA from nucleolytic degradation.

    Evidence DNA fiber and nascent-DNA degradation assays with RNF169 depletion in human cells

    PMID:41145912

    Open questions at the time
    • Single lab, not yet independently replicated
    • The protective effectors RNF169 recruits at forks are not identified
    • Relationship between the fork role and the DSB role is unclear

Open questions

Synthesis pass · forward-looking unresolved questions
  • How RNF169's competitive readout, USP7-dependent stability, DYRK1A phosphorylation, and replication-fork reader activity are integrated to govern repair-pathway choice in a cell-cycle- and lesion-specific manner remains unresolved.
  • No unified model linking DSB and replication-fork functions
  • Physiological and disease contexts requiring RNF169 are uncharacterized in the corpus

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0042393 histone binding 3 GO:0060089 molecular transducer activity 2 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0000228 nuclear chromosome 3 GO:0005634 nucleus 1
Pathway
R-HSA-73894 DNA Repair 3 R-HSA-4839726 Chromatin organization 1 R-HSA-69306 DNA Replication 1
Partners
DYRK1ARNF168TP53BP1USP7

Evidence

Reading pass · 7 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2012 RNF169 accumulates at DNA double-strand break (DSB) repair foci through recognition of RNF168-catalyzed ubiquitylation products via its motif interacting with ubiquitin (MIU) domain, in an RNF8/RNF168-dependent manner. Cellular localization assays, domain mapping, loss-of-function experiments in human cells The Journal of cell biology High 22492721 22733822
2012 RNF169 competitively limits the recruitment of 53BP1 and RAP80-BRCA1 to RNF168-modified chromatin at DSB sites, independent of its own catalytic E3 ligase activity, thereby stimulating homologous recombination and restraining non-homologous end joining. Overexpression and knockdown in human cells, competition assays for DSB foci accumulation, cell survival assays after DSB infliction The Journal of cell biology High 22492721 22733822
2017 RNF169 binds ubiquitylated H2A-Lys13/Lys15 on the nucleosome through a three-pronged interaction: its canonical ubiquitin-binding helix plus two arginine-rich motifs that contact the nucleosome acidic patch, as revealed at atomic resolution. Methyl-TROSY solution NMR spectroscopy, molecular dynamics simulations, electron cryo-microscopy (cryo-EM), mutagenesis validation eLife High 28406400
2017 RNF169 contains a dual-function nuclear localization signal (NLS) that both imports RNF169 into the nucleus and mediates a direct interaction with the deubiquitylase USP7, which stabilizes RNF169; disrupting the USP7–RNF169 interaction destabilizes RNF169, compromises high-fidelity DSB repair, and hypersensitizes cells to PARP inhibition. Crystal structure of USP7 bound to RNF169 NLS peptide, structure-guided mutagenesis to uncouple USP7 binding from nuclear import, cellular stability and DSB repair assays Proceedings of the National Academy of Sciences of the United States of America High 28325877
2018 RNF169 preferentially accumulates at DNA end-proximal, resected (RPA-bound) DSBs, promotes CtIP-dependent DSB end resection, and stimulates single-strand annealing repair dose-dependently by alleviating the 53BP1-imposed barrier to resection. ChIP analyses of AsiSI-induced DSBs, RNF169 knockdown/overexpression, single-strand annealing reporter assays, epistasis with CtIP and 53BP1 Proceedings of the National Academy of Sciences of the United States of America High 30104380
2019 DYRK1A kinase directly phosphorylates RNF169 at two sites; phosphorylation of at least one site enhances RNF169's ability to displace 53BP1 from DSB sites and promotes homologous recombination repair. DYRK1A overexpression inhibits 53BP1 accumulation at DSBs in an RNF169-dependent manner. Proteomic affinity purification-mass spectrometry, in vitro kinase assay, phosphosite mutagenesis, overexpression/CRISPR-KO in U-2 OS cells, 53BP1 foci quantification Cell cycle (Georgetown, Tex.) / Scientific reports Medium 30773093 30979931
2025 RNF169 functions as a reader of the H2BK120ub histone mark at stalled replication forks, where it is required to protect nascent DNA from excessive nucleolytic degradation; loss of RNF169 at stalled forks leads to defective protection of nascent DNA. Replication fork assays (DNA fiber), nascent DNA degradation assays, localization of RNF169 to replication forks, loss-of-function by depletion in human cells The EMBO journal Medium 41145912

Source papers

Stage 0 corpus · 9 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2012 Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks. The Journal of cell biology 121 22492721
2012 Ring finger protein RNF169 antagonizes the ubiquitin-dependent signaling cascade at sites of DNA damage. The Journal of biological chemistry 63 22733822
2017 Dual-utility NLS drives RNF169-dependent DNA damage responses. Proceedings of the National Academy of Sciences of the United States of America 58 28325877
2017 The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage. eLife 48 28406400
2018 RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair. Proceedings of the National Academy of Sciences of the United States of America 45 30104380
2019 DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169. Cell cycle (Georgetown, Tex.) 41 30773093
2019 A comprehensive proteomics-based interaction screen that links DYRK1A to RNF169 and to the DNA damage response. Scientific reports 38 30979931
2019 An LTR retrotransposon-derived lncRNA interacts with RNF169 to promote homologous recombination. EMBO reports 30 31486214
2025 H2BK120ub and its reader RNF169 sequentially regulate replication fork remodeling and stability. The EMBO journal 3 41145912

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