Affinage

ZRANB3

DNA annealing helicase and endonuclease ZRANB3 · UniProt Q5FWF4

Length
1079 aa
Mass
123.2 kDa
Annotated
2026-06-11
27 papers in source corpus 14 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ZRANB3 is an SNF2-family ATP-dependent DNA translocase that acts at stalled and damaged replication forks to enforce error-free DNA damage tolerance and genomic stability (PMID:22704558, PMID:28886337). It is recruited to arrested forks through coincident recognition of K63-linked polyubiquitinated PCNA, the replication machinery, and branched DNA structures, and its depletion elevates sister chromatid exchange and sensitivity to replication stress (PMID:22704558, PMID:22759634). Mechanistically, ZRANB3 catalyzes replication fork reversal in a manner that requires its translocase activity and UBC13-dependent PCNA ubiquitination, with loss of this activity causing unrestrained fork progression and chromosomal breakage (PMID:28886337). Beyond fork reversal it functions as an annealing helicase that rewinds RPA-bound complementary ssDNA via a HARP-like (HPL) domain, and carries a separable, structure-specific ATP-dependent endonuclease activity that cleaves branched DNA with unusual polarity to generate an accessible leading-strand 3'-OH (PMID:22705370, PMID:22759634, PMID:21078962); substrate recognition of forked DNA, ATP hydrolysis, and these catalytic activities depend on a dedicated substrate recognition domain analogous to accessory domains in SMARCAL1 and HLTF (PMID:26884333). Its motor-driven activities are directly stimulated by RAD51 and the BCDX2 paralog complex, and within the fork-remodeling enzyme set ZRANB3 is particularly efficient at branch migration downstream of fork reversal (PMID:35801922). ZRANB3 is recruited and its remodeling activity promoted by the ubiquitin ligase RFWD3, which enhances PCNA ubiquitination and the PCNA-ZRANB3 interaction (PMID:37036693), while nuclear RNR-α hexamers directly bind and inhibit ZRANB3-promoted DNA synthesis (PMID:30150681). At the organismal level, ZRANB3 supports long-term hematopoietic stem cell function and limits replication-stress-driven damage (PMID:39044358), contributes to pancreatic β-cell survival and glucose-stimulated insulin secretion (PMID:31324766), and has distinct, non-redundant in vivo roles from SMARCAL1 in resolving oncogene-induced replication stress (PMID:30610086).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2010 High

    Established the core biochemical identity of the protein by asking what enzymatic activity it carries, showing it is an annealing helicase distinct from SMARCAL1.

    Evidence In vitro ATPase, strand-annealing, helicase (negative), and RPA-interaction (negative) assays with purified recombinant AH2/ZRANB3

    PMID:21078962

    Open questions at the time
    • No cellular function assigned at this stage
    • HNH/nuclease activity not observed under the conditions tested
    • No recruitment mechanism identified
  2. 2012 High

    Defined how ZRANB3 is targeted to and acts at stalled forks, answering both recruitment (polyubiquitinated PCNA, branched DNA) and activity (fork remodeling, recombination intermediate disassembly, structure-specific endonuclease).

    Evidence Reciprocal Co-IP, ubiquitin-chain and DNA-binding assays, in vitro remodeling and endonuclease assays, domain mutagenesis, and cellular knockdown with SCE/damage readouts across three concurrent papers

    PMID:22704558 PMID:22705370 PMID:22759634

    Open questions at the time
    • Did not establish whether fork reversal occurs in cells
    • In vivo regulators of recruitment not identified
    • Physiological substrates of the endonuclease at endogenous forks not defined
  3. 2016 High

    Localized substrate recognition to a discrete domain, explaining how the enzyme couples forked-DNA binding to ATP hydrolysis and catalysis.

    Evidence Domain deletion/mutagenesis with in vitro ATPase, fork remodeling, and endonuclease assays on recombinant domain mutants

    PMID:26884333

    Open questions at the time
    • No structural model of the SRD-DNA interface
    • Single lab
  4. 2017 High

    Demonstrated that ZRANB3 translocase activity drives damage-induced fork reversal in cells, linking the in vitro motor to a defined replication intermediate and to genome stability.

    Evidence Electron microscopy of replication intermediates with translocase-dead mutants, DNA fiber assays, and UBC13 depletion epistasis

    PMID:28886337

    Open questions at the time
    • Relative contributions of ZRANB3 versus SMARCAL1/HLTF to reversal not fully resolved
    • How reversal is reversed/restarted not addressed here
  5. 2018 Medium

    Identified a direct negative regulator, showing nuclear RNR-α hexamers bind ZRANB3 and suppress its promotion of DNA synthesis in unstressed cells.

    Evidence Reciprocal Co-IP, nuclear fractionation, DNA synthesis assays with ZRANB3 knockdown/knockout, and hexamerization-inducing nucleotide analogs

    PMID:30150681

    Open questions at the time
    • Structural basis of RNR-α–ZRANB3 inhibition not defined
    • Single lab
    • Whether inhibition targets translocase or endonuclease activity unclear
  6. 2019 Medium

    Connected ZRANB3 to disease-relevant contexts: DNA synthesis and transformation, drug response to RNR-targeting dATP analogs, oncogene-induced replication stress, and β-cell biology.

    Evidence ZRANB3 KO/KD with DNA synthesis and drug-resistance assays and H-rasG12V transformation; mouse Smarcal1/Zranb3 haploinsufficiency lymphoma models; zebrafish and MIN6 β-cell loss-of-function with apoptosis and insulin secretion readouts

    PMID:30610086 PMID:31324766 PMID:31836351

    Open questions at the time
    • Molecular basis for opposing Smarcal1/Zranb3 effects on lymphomagenesis unresolved
    • Mechanism linking ZRANB3 to β-cell survival/insulin secretion not defined
    • Single lab per system
  7. 2022 High

    Showed that ZRANB3's motor activity is positively regulated by recombination factors, with RAD51 and BCDX2 directly stimulating its ATPase/translocase and defining its specialization in branch migration.

    Evidence Reconstituted in vitro reactions with purified proteins, ATPase, fork reversal, branch migration assays, and pull-downs for physical interactions

    PMID:35801922

    Open questions at the time
    • Stimulation not yet validated at endogenous forks in cells
    • Structural mechanism of stimulation unknown
    • Single lab
  8. 2023 High

    Identified RFWD3 as an upstream ligase that promotes ZRANB3 recruitment and fork remodeling by enhancing PCNA ubiquitination and the PCNA-ZRANB3 interaction.

    Evidence Electron microscopy of replication intermediates, Co-IP, KO/KD epistasis, DNA fiber and PCNA ubiquitination assays, nuclear foci localization

    PMID:37036693

    Open questions at the time
    • Direct ubiquitination targets of RFWD3 versus UBC13 contributions not fully separated
    • Single lab
  9. 2024 Medium

    Extended ZRANB3 function to stem cell maintenance, showing it preserves long-term HSC function and limits age-dependent replication-stress-driven hematopoietic dysregulation.

    Evidence Mouse knockout, bone marrow transplantation, DNA fiber assays, γH2AX staining, and HSPC flow cytometry/aging studies

    PMID:39044358

    Open questions at the time
    • Cell-intrinsic molecular pathway in HSCs not dissected
    • How non-redundancy with Smarcal1 is achieved unclear
    • Single lab

Open questions

Synthesis pass · forward-looking unresolved questions
  • How ZRANB3's distinct activities (fork reversal, branch migration, endonuclease) are selected and switched in vivo, and how chromatin context such as H2B monoubiquitination gates its activity, remains unresolved.
  • No structural model of ZRANB3 engaging a fork
  • Chromatin-level regulation only addressed in a single preprint co-depletion study (PMID-less bioRxiv)
  • Switch between translocase and endonuclease modes at endogenous forks undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140657 ATP-dependent activity 5 GO:0003677 DNA binding 3 GO:0140097 catalytic activity, acting on DNA 2 GO:0016787 hydrolase activity 1
Localization
GO:0000228 nuclear chromosome 2 GO:0005634 nucleus 2
Pathway
R-HSA-73894 DNA Repair 3 R-HSA-69306 DNA Replication 2
Partners
PCNARAD51RAD51BRAD51CRAD51DRFWD3RRM1XRCC2

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2012 ZRANB3 is recruited to stalled replication forks via interaction with polyubiquitinated PCNA (K63-linked polyubiquitin chains), promoting fork restart following replication arrest. ZRANB3 depletion increases sister chromatid exchange and DNA damage sensitivity after replication stress. In vitro biochemical assays with recombinant ZRANB3, co-immunoprecipitation, mammalian cell knockdown with phenotypic readouts (SCE frequency, DNA damage sensitivity) Molecular cell High 22704558
2012 Recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates in vitro, consistent with a role in limiting inappropriate recombination during template switching. In vitro biochemical assays with recombinant ZRANB3 on synthetic DNA substrates mimicking stalled forks and recombination intermediates Molecular cell High 22704558
2012 AH2/ZRANB3 contains a HARP-like (HPL) domain that is indispensable for its annealing activity in vitro and its function in vivo at stalled replication forks. AH2 binds PCNA, which is crucial for its recruitment to stalled forks. Domain deletion/mutation analysis, in vitro annealing assays, Co-IP with PCNA, cellular knockdown with replication stress sensitivity assays Molecular cell High 22705370
2012 ZRANB3 possesses a structure-specific ATP-dependent endonuclease activity that cleaves branched DNA structures with unusual polarity, generating an accessible 3'-OH group in the template of the leading strand. This endonuclease activity is coupled to ATP hydrolysis. In vitro endonuclease assays with recombinant ZRANB3 on branched DNA substrates; mutagenesis to separate translocase from endonuclease activities Genes & development High 22759634
2012 ZRANB3 localizes to DNA replication sites and interacts with components of the replication machinery. It is recruited to damaged replication forks via multiple mechanisms: interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures. Immunofluorescence co-localization with replication markers, Co-IP with PCNA and ubiquitin chain pull-downs, in vitro DNA-binding assays Genes & development High 22759634
2010 AH2/ZRANB3 is an annealing helicase that catalyzes ATP-dependent rewinding of RPA-bound complementary single-stranded DNA but does not exhibit detectable helicase (unwinding) activity. Unlike SMARCAL1/HARP, AH2 lacks a conserved RPA-binding domain and does not interact with RPA. AH2 contains an HNH motif but purified AH2 does not exhibit nuclease activity under standard conditions. In vitro ATPase assay, strand-annealing/rewinding assay with RPA-coated ssDNA, helicase assay (negative result for unwinding), RPA interaction assay (negative result) Proceedings of the National Academy of Sciences of the United States of America High 21078962
2016 A substrate recognition domain (SRD) within ZRANB3 is required for recognition of forked DNA structures, ATP hydrolysis, catalysis of fork remodeling, and structure-specific endonuclease activity. This domain is analogous to accessory substrate-binding domains in related SNF2 enzymes SMARCAL1 and HLTF. Domain deletion/mutagenesis, in vitro ATPase assays, fork remodeling assays, endonuclease assays with recombinant ZRANB3 domain mutants The Journal of biological chemistry High 26884333
2017 Damage-induced replication fork reversal in mammalian cells requires PCNA ubiquitination (K63-linked polyubiquitin chains via UBC13), ZRANB3 translocase activity, and its interaction with polyubiquitinated PCNA. Mutations abolishing fork reversal caused unrestrained fork progression and chromosomal breakage. Electron microscopy of replication intermediates, DNA fiber assays, ZRANB3 translocase-dead mutants, UBC13 depletion, cellular DNA damage assays Molecular cell High 28886337
2018 Nuclear RNR-α directly interacts with ZRANB3 and suppresses ZRANB3's function in promoting DNA synthesis in unstressed cells. RNR-α hexamerization (induced by dA-analog nucleotides) promotes RNR-α nuclear import and subsequent ZRANB3 inhibition. Co-immunoprecipitation of RNR-α with ZRANB3, nuclear fractionation, DNA synthesis assays with ZRANB3 knockdown/knockout, RNR-α hexamerization-inducing nucleotide analogs Nature chemical biology Medium 30150681
2019 ZRANB3 promotes DNA synthesis in cells and plays a role in tumor invasion and H-rasG12V-promoted transformation. Anti-leukemic RNR-inhibiting dATP-analogs (e.g., clofarabine) target ZRANB3 to inhibit DNA synthesis; ZRANB3 knockout/knockdown confers resistance to these drugs. ZRANB3 knockdown/knockout cells, DNA synthesis assays, drug resistance assays, H-rasG12V transformation assay Cell chemical biology Medium 31836351
2022 RAD51 and the RAD51 paralog complex BCDX2 (RAD51B-RAD51C-RAD51D-XRCC2) directly stimulate the motor-driven (ATPase translocase) activities of ZRANB3 in reconstituted reactions, underpinned by physical interactions between ZRANB3 and RAD51/BCDX2. ZRANB3 (with HLTF but not SMARCAL1) is efficient in branch migration downstream of fork reversal, whereas SMARCAL1 (with ZRANB3 but not HLTF) efficiently rezips RPA-covered bubbled DNA. Reconstituted in vitro biochemical assays with purified recombinant proteins, ATPase assays, fork reversal assays, branch migration assays, pull-down for physical interactions Nucleic acids research High 35801922
2023 The ubiquitin ligase RFWD3 promotes recruitment of ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner, promotes PCNA ubiquitination, and enhances PCNA-ZRANB3 interaction, providing a mechanism for RFWD3-dependent ZRANB3 recruitment. Electron microscopy of replication intermediates, Co-IP, ZRANB3 and RFWD3 knockdown/knockout cells, DNA fiber assays, PCNA ubiquitination assays, nuclear foci localization The Journal of cell biology High 37036693
2019 Smarcal1 and Zranb3 have nonredundant functions in resolving Myc-induced DNA replication stress in primary cells. Haploinsufficiency of Smarcal1 accelerates Myc-induced lymphomagenesis, whereas haploinsufficiency of Zranb3 inhibits lymphoma development, demonstrating distinct in vivo roles despite related biochemical functions. Mouse genetic models (haploinsufficiency, complete loss), DNA fiber assays, γH2AX staining, lymphoma development assays, apoptosis and proliferation assays Cancer research Medium 30610086
2024 Loss of Zranb3 in mice causes defective long-term hematopoietic stem cell (HSC) function revealed by bone marrow transplantation, and age-dependent acceleration of myeloid-biased hematopoietic dysregulation driven by accumulated DNA damage and replication stress. Zranb3 and Smarcal1 have distinct, non-redundant roles in different hematopoietic stem and progenitor cell populations. Mouse knockout models, bone marrow transplantation, DNA fiber assays, γH2AX staining, HSPC flow cytometry, aging studies Aging cell Medium 39044358
2019 Knockdown or knockout of the zebrafish ortholog of ZRANB3 reduces pancreatic β-cell number due to increased apoptosis in islets. siRNA knockdown of murine Zranb3 in MIN6 β-cells impairs insulin secretion in response to high glucose, implicating Zranb3 in β-cell functional response. Zebrafish morpholino knockdown and CRISPR knockout, immunofluorescence for β-cell number, apoptosis assays, murine MIN6 cell siRNA knockdown with glucose-stimulated insulin secretion assay Nature communications Medium 31324766
2024 RNF20-mediated H2B monoubiquitination protects stalled replication forks from MRE11-dependent degradation; co-depletion of ZRANB3 (along with SMARCAL1 and HLTF) rescues fork degradation in RNF20-depleted cells, placing ZRANB3 as a fork remodeler whose activity becomes detrimental without H2Bub-mediated fork protection. siRNA co-depletion epistasis, DNA fiber assays, MRE11 inhibition, RNF20 mutant analysis bioRxivpreprint Low bio_10.1101_2024.11.25.625131

Source papers

Stage 0 corpus · 27 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2012 Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress. Molecular cell 249 22704558
2017 Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity. Molecular cell 204 28886337
2017 Functions of SMARCAL1, ZRANB3, and HLTF in maintaining genome stability. Critical reviews in biochemistry and molecular biology 124 28954549
2012 The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress. Molecular cell 104 22705370
2012 ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response. Genes & development 100 22759634
2019 ZRANB3 is an African-specific type 2 diabetes locus associated with beta-cell mass and insulin response. Nature communications 81 31324766
2010 Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B. Journal of virology 67 20926561
1981 The pyridine nucleotide cycle. Studies in Escherichia coli and the human cell line D98/AH2. The Journal of biological chemistry 51 7021549
2010 Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif. Proceedings of the National Academy of Sciences of the United States of America 45 21078962
2019 AH2 encodes a MYB domain protein that determines hull fate and affects grain yield and quality in rice. The Plant journal : for cell and molecular biology 36 31357245
2022 Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex. Nucleic acids research 34 35801922
2019 Smarcal1 and Zranb3 Protect Replication Forks from Myc-Induced DNA Replication Stress. Cancer research 29 30610086
2003 Elucidation of the Vibrio anguillarum genetic response to the potential fish probiont Pseudomonas fluorescens AH2, using RNA-arbitrarily primed PCR. Journal of bacteriology 25 12533458
2018 Nuclear RNR-α antagonizes cell proliferation by directly inhibiting ZRANB3. Nature chemical biology 23 30150681
2016 Identification of a Substrate Recognition Domain in the Replication Stress Response Protein Zinc Finger Ran-binding Domain-containing Protein 3 (ZRANB3). The Journal of biological chemistry 22 26884333
2023 RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks. The Journal of cell biology 21 37036693
2015 Interaction between the NS4B amphipathic helix, AH2, and charged lipid headgroups alters membrane morphology and AH2 oligomeric state--Implications for the Hepatitis C virus life cycle. Biochimica et biophysica acta 15 25944559
2023 Lactobacillus paracasei AH2 isolated from Chinese sourdough alleviated gluten-induced food allergy through modulating gut microbiota and promoting short-chain fatty acid accumulation in a BALB/c mouse model. Journal of the science of food and agriculture 10 37653286
2019 Clofarabine Commandeers the RNR-α-ZRANB3 Nuclear Signaling Axis. Cell chemical biology 9 31836351
2013 N-terminal AH2 segment of protein NS4B from hepatitis C virus. Binding to and interaction with model biomembranes. Biochimica et biophysica acta 9 23639583
2017 Development of a realtime RT-PCR assay for the rapid detection of influenza A(H2) viruses. Molecular and cellular probes 4 28652020
2024 DNA fork remodeling proteins, Zranb3 and Smarcal1, are uniquely essential for aging hematopoiesis. Aging cell 3 39044358
2026 The Zinc Finger Ran-Binding Protein 3 (ZRANB3): An Advanced Perspective. International journal of molecular sciences 1 41596226
2025 Australian Hard 2 (AH2) wheat class with null4A genetic basis to produce high-quality Japanese ramen noodle. Journal of the science of food and agriculture 1 41261959
2024 WITHDRAWN: Strand dependent bypass of DNA lesions during fork reversal by ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF. bioRxiv : the preprint server for biology 0 39345618
2022 Draft Genome Sequence of Streptococcus agalactiae Strain AH2, Isolated from Hybrid Tilapia (Oreochromis niloticus × Oreochromis aureus). Microbiology resource announcements 0 36350140
2016 [The development of immune enzyme test-systems for detecting potentially pandemic influenza viruses subtype A(H2), A(H5), A(H7) and A(H9).]. Klinicheskaia laboratornaia diagnostika 0 31529925

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