Affinage

APLF

Aprataxin and PNK-like factor · UniProt Q8IW19

Length
511 aa
Mass
57.0 kDa
Annotated
2026-06-09
26 papers in source corpus 21 papers cited in narrative 21 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

APLF is a multifunctional scaffold and histone chaperone that operates at DNA strand breaks to coordinate non-homologous end-joining (NHEJ) and restore chromatin (PMID:17353262, PMID:23178593). It is recruited to damage sites by two parallel routes: an FHA-domain interaction that recognizes CK2-phosphorylated motifs in XRCC1 and XRCC4 (PMID:17353262, PMID:18077224, PMID:29059378), and a PAR-dependent route mediated by its C-terminal tandem PBZ zinc fingers, which bind poly(ADP-ribose) through conserved aromatic and basic residues and accumulate at breaks downstream of PARP-3 signaling (PMID:18474613, PMID:20439749, PMID:21211721). Once recruited, APLF acts as an intrinsically disordered scaffold that bridges Ku80 — through a discrete Ku-binding motif (KBM) that docks on the Ku80 alpha/beta domain at a site distinct from the FHA interaction — with XRCC4-DNA Ligase IV and XLF, assembling and stabilizing the NHEJ ligation complex and supporting DNA end synapsis under force (PMID:23178593, PMID:23689425, PMID:30291363, PMID:36640344). In parallel, its C-terminal acidic domain functions as a histone chaperone that binds both the (H3-H4)2 tetramer and H2A-H2B and can assemble and deposit the complete histone octamer in a single step to reform nucleosomes, linking ligation to chromatin restoration (PMID:21211722, PMID:29905837, PMID:35895815). APLF is phosphorylated at Ser-116 by ATM in a PARP-3- and PBZ-dependent manner to promote DSB repair (PMID:17507382, PMID:23449221), and loss of APLF biases class switch recombination toward microhomology-mediated end-joining (PMID:21211721). Beyond canonical NHEJ, PARP1-dependent PBZ recruitment positions APLF at stalled replication forks, where it promotes FANCD2 loading and protects nascent DNA during interstrand crosslink repair (PMID:38520407).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2007 High

    Established APLF as a DNA-damage-responsive factor by showing it accumulates at breaks through two independent routes and is required for efficient strand break repair, defining its core role in the repair response.

    Evidence Yeast two-hybrid, reciprocal co-IP, YFP live imaging, and siRNA repair kinetics across two concurrent labs identifying FHA-XRCC1 and C-terminal zinc finger recruitment mechanisms

    PMID:17353262 PMID:17507382

    Open questions at the time
    • The PAR ligand of the zinc finger route was not yet identified
    • The catalytic outcome of XRCC1 binding (transport vs. retention) was unresolved
  2. 2008 High

    Identified the ligand and partners of the two recruitment arms, showing the C-terminal zinc fingers bind PAR while the FHA domain engages CK2-phosphorylated XRCC4-Lig4, and APLF associates with Ku at DNA ends — placing APLF directly within NHEJ.

    Evidence PAR-binding and in vitro PAR synthesis assays, in vitro CK2 kinase assay, co-IP, and NHEJ reporter assays

    PMID:18077224 PMID:18474613

    Open questions at the time
    • Structural basis of PBZ-PAR recognition not yet defined
    • How APLF physically bridges Ku and XRCC4-Lig4 not yet mapped
  3. 2010 High

    Resolved the structural basis of PAR recognition, showing the PBZ modules form a novel zinc finger fold whose conserved aromatic and basic residues contact ADP-ribose and are required for damage recruitment.

    Evidence NMR solution structures, crystallographic analysis, site-directed mutagenesis, and in vivo recruitment assays

    PMID:20098424 PMID:20439749

    Open questions at the time
    • Did not establish how PAR binding is coupled to downstream complex assembly
    • The upstream PARP responsible in vivo was not yet defined
  4. 2011 High

    Defined the upstream signal and dual function of APLF, placing PARP-3 upstream of APLF recruitment and showing APLF both retains XRCC4-Lig4 in chromatin and acts as a DNA-damage-specific histone chaperone via its acidic NAP1L-like domain.

    Evidence PARP-3 stimulation and epistasis assays, Aplf-/- B cell class switch recombination, chromatin fractionation, and in vitro histone chaperone reconstitution

    PMID:21211721 PMID:21211722

    Open questions at the time
    • Histone substrate specificity (H3/H4 vs. H2A/H2B vs. octamer) not fully resolved
    • Mechanism coupling ligation to chromatin restoration unclear
  5. 2012 High

    Established APLF as the scaffold that nucleates the Ku-anchored ligation complex, showing the Ku80 vWA domain recruits APLF, which in turn promotes assembly of XRCC4-Lig4 and XLF into a ligation-competent complex.

    Evidence Co-IP, domain mapping, in vitro DNA ligation reconstitution, and NHEJ reporter/clonogenic assays in DT40 and human cells

    PMID:23178593

    Open questions at the time
    • Precise Ku80 docking site for APLF not yet defined at atomic resolution
    • Stoichiometry within the assembled complex unresolved
  6. 2013 Medium

    Refined the recruitment and signaling logic by defining a discrete Ku-binding motif (KBM) distinct from the FHA interaction and showing ATM-mediated Ser-116 phosphorylation depends on PARP3 and the PBZ domains.

    Evidence Peptide reconstitution, mutagenesis, immunofluorescence at laser/IR damage, S116A phosphomutant analysis, and ATM/PARP3 depletion

    PMID:23449221 PMID:23689425

    Open questions at the time
    • No in vitro reconstitution of the ATM-PARP3-APLF signaling axis
    • Functional consequence of Ser-116 phosphorylation on complex assembly not isolated
  7. 2017 Medium

    Provided the atomic and dynamic picture of APLF within NHEJ, showing it is intrinsically disordered and uses its flexibility to tether Ku/DNA-PK and XRCC4-Lig4 into an extended six-protein core complex, with the FHA-phospho-XRCC1 interaction structurally defined.

    Evidence SAXS with mutagenesis and in vitro complex assembly; X-ray crystallography, NMR, and fluorescence polarization of the FHA-XRCC1 complex

    PMID:27875301 PMID:29059378

    Open questions at the time
    • Dynamics of the assembled complex during ligation not directly visualized
    • pH-dependence of FHA binding in cells not established
  8. 2018 High

    Resolved the dual histone chaperone activity and Ku-docking architecture, showing the acidic domain anchors both H2A-H2B and H3-H4 via aromatic side chains, and that APLF and XLF KBMs bind remote, independent Ku80 sites to recruit their partners.

    Evidence NMR and crystal structures, histone binding/chaperone assays, mutagenesis, laser microirradiation, and end-joining/radiosensitivity assays

    PMID:29905837 PMID:30291363

    Open questions at the time
    • How a single acidic domain handles full octamer not yet shown
    • Coordination between Ku-scaffolding and histone chaperone functions unresolved
  9. 2022 High

    Unified the chaperone and synapsis functions, showing the acidic domain assembles and deposits the complete histone octamer in one step and that APLF stabilizes DNA end bridging within a minimal Ku-APLF synaptic complex.

    Evidence Crystal structure of the APLFAD-octamer complex, in vitro nucleosome reconstitution, and magnetic-tweezers single-molecule synapsis assays with domain deletions

    PMID:35895815 PMID:36640344

    Open questions at the time
    • In vivo timing of octamer deposition relative to ligation not defined
    • Regulation of the acidic domain's switch between bridging and chromatin assembly unclear
  10. 2024 Medium

    Extended APLF function beyond DSBs to replication stress, showing PARP1-dependent PBZ recruitment to stalled forks enables FANCD2 loading and nascent-strand protection during ICL repair.

    Evidence siRNA depletion, DNA fiber fork-protection assays, co-IP, FANCD2/APLF immunofluorescence, PARP1 inhibition, and cisplatin sensitivity

    PMID:38520407

    Open questions at the time
    • Direct APLF-FANCD2 interaction vs. indirect recruitment not distinguished
    • Single lab; mechanism of fork-localized protection not reconstituted
  11. 2024 Low

    Reported candidate non-canonical roles in centrosome regulation and EMT-linked nuclear localization, proposing intrinsic kinase activity and PARP1-driven nuclear transport.

    Evidence Immunofluorescence, enzymatic kinase and docking assays in mouse ESCs; subcellular fractionation, NLS-tagged APLF, PARP1 inhibition, and invasion assays in breast cancer cells

    PMID:38968704 PMID:39384105

    Open questions at the time
    • Kinase activity lacks in vitro reconstitution with defined substrate and rigorous controls; requires independent validation
    • PARP1-mediated nuclear transport shown only indirectly without co-transport reconstitution
    • Centrosomal and EMT roles not connected to the established DNA repair functions

Open questions

Synthesis pass · forward-looking unresolved questions
  • How APLF's distinct activities — Ku scaffolding, end synapsis, histone octamer chaperoning, and fork protection — are temporally coordinated and regulated at a single break or fork remains unresolved.
  • No integrated model of the order of synapsis, ligation, and chromatin restoration
  • Regulatory switch governing the acidic domain's dual bridging/chaperone roles unknown
  • In vivo significance of non-repair roles (centrosome, EMT) unestablished

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0042393 histone binding 3 GO:0060090 molecular adaptor activity 3 GO:0003677 DNA binding 1
Localization
GO:0005634 nucleus 3 GO:0000228 nuclear chromosome 2
Pathway
R-HSA-4839726 Chromatin organization 3 R-HSA-73894 DNA Repair 3
Partners
FANCD2LIG4PARP3XLFXRCC1XRCC4XRCC5XRCC6
Complex memberships
NHEJ core complex (Ku-APLF-XRCC4-Lig4-XLF)

Evidence

Reading pass · 21 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2007 APLF (C2orf13) accumulates at sites of chromosomal DNA damage via two distinct mechanisms: (1) an FHA domain-mediated interaction with XRCC1 (stimulated by CK2 phosphorylation of XRCC1) and (2) an FHA-independent mechanism requiring a C-terminal zinc finger motif. APLF is also phosphorylated in a DNA damage- and ATM-dependent manner, and its depletion reduces rates of chromosomal DNA strand break repair. Yeast two-hybrid, in vitro and in vivo co-IP, YFP-tagging with live imaging, siRNA knockdown with repair kinetics assay Molecular and cellular biology High 17353262
2007 Xip1/APLF interacts with XRCC1 through recognition of CK2 phosphorylation sites in XRCC1 by the FHA domain of Xip1, and XRCC1 is required to maintain steady-state levels of Xip1. Xip1 is phosphorylated on Ser-116 by ATM in response to ionizing radiation. The C-terminal zinc finger motif is required for recruitment to DNA break sites independently of XRCC1. Co-IP, GFP live-cell imaging, PARP-1 inhibition, siRNA knockdown, clonogenic survival assay The Journal of biological chemistry High 17507382
2008 The C-terminal tandem zinc finger domain of APLF binds tightly to poly(ADP-ribose) (PAR), enabling PAR-dependent accumulation at sites of chromosomal damage. APLF negatively affects poly(ADP-ribosylation) in vitro in a zinc finger-dependent manner, and overexpression of APLF or its C-terminal zinc finger fragment suppresses PAR appearance in human cells. PAR-binding assay, overexpression in human A549 cells, zinc finger mutant analysis, in vitro PAR synthesis assay Molecular and cellular biology High 18474613
2008 APLF interacts with Ku and XRCC4-DNA ligase IV in human cells. The interaction with XRCC4-DNA ligase IV is FHA-domain- and phospho-dependent, mediated by CK2 phosphorylation of XRCC4 in vitro. The interaction with Ku is independent of the FHA and zinc finger domains, and APLF associates with Ku at DNA ends. ATM phosphorylates APLF at Ser-116 following IR. Depletion of APLF by siRNA impairs NHEJ. Co-IP, in vitro kinase assay, siRNA knockdown, NHEJ reporter assay, phospho-specific antibody DNA repair High 18077224
2010 NMR solution structures of the two PBZ (PAR-binding zinc finger) modules of APLF reveal a novel type of zinc finger. In vivo PAR-binding and NMR interaction data with PAR fragments provide a structural basis for PBZ-PAR recognition. NMR spectroscopy, in vivo PAR-binding assay Nature structural & molecular biology High 20098424
2010 Conserved residues Y381/Y386 and Y423/Y428 in the C(M/P)Y and CYR motifs within each APLF PBZ domain are critical for interaction with the adenine ring of ADP-ribose, while basic residues R387 and R429 coordinate interactions with the phosphate backbone. These residues are required for APLF recruitment to sites of DNA damage in vivo. Crystallography/structural analysis, biochemical binding assay, site-directed mutagenesis, in vivo recruitment assay Proceedings of the National Academy of Sciences of the United States of America High 20439749
2011 PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro, functions in the same pathway as APLF (genetic epistasis), and is required for APLF accumulation at DSBs. APLF promotes retention of the XRCC4/DNA ligase IV complex in chromatin. In Aplf-/- B cells, class switch recombination is biased toward microhomology-mediated end-joining; overexpression of XRCC4/DNA ligase IV circumvents the requirement for both PARP-3 and APLF. In vitro PARP-3 stimulation assay, co-IP, chromatin fractionation, Aplf-/- mouse B cell class switch recombination assay, XRCC4/LigIV overexpression epistasis Molecular cell High 21211721
2011 APLF is a DNA-damage-specific histone chaperone that preferentially binds the histone H3/H4 tetramer via its C-terminal acidic domain, which contains a NAP1L motif homologous to NAP1L family chaperones. The acidic domain is required for histone chaperone activity in vitro and for APLF repair capacity in vivo. In vitro histone binding/chaperone assay, mutational analysis of acidic domain, in vivo DNA repair assay, pulldown Molecular cell High 21211722
2012 The von Willebrand (vWA) domain of Ku80 recruits APLF into Ku-DNA complexes. APLF functions as a scaffold protein promoting recruitment and/or retention of XRCC4-Lig4 and XLF, assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of APLF–Ku80 or APLF–XRCC4-Lig4 interactions impairs NHEJ and confers cellular hypersensitivity. Co-IP, in vitro DNA ligation assay, domain mapping, mutagenesis, DT40 and human cell NHEJ reporter assays, clonogenic survival The EMBO journal High 23178593
2013 ATM phosphorylation of APLF at Ser-116 is dependent on PARP3 levels and the APLF PBZ domains. Depletion or inhibition of ATM or PARP3 reduces APLF accumulation at DNA damage sites and impairs DSB repair kinetics. ATM and PARP3 operate in a common signaling pathway leading to APLF-Ser-116 phosphorylation. siRNA depletion, chemical inhibition of ATM/PARP3, phospho-APLF immunofluorescence at laser-induced damage/IR-induced foci, phosphomutant (S116A) analysis Nucleic acids research Medium 23449221
2013 A conserved Ku-binding motif (KBM) within APLF is required for the physical interaction with Ku, distinct from the FHA-domain interaction with XRCC4. Disruption of the KBM increases cytoplasmic relocalization of APLF and reduces XRCC4 association; introduction of an NLS rescues nuclear localization. Both Ku-binding and FHA-XRCC4 interactions are required for efficient NHEJ and APLF retention at damage sites. Domain mapping, in vitro peptide reconstitution, mutagenesis, immunofluorescence, NHEJ reporter assay in APLF-depleted cells reconstituted with mutants The Journal of biological chemistry Medium 23689425
2016 SAXS and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and XRCC4-DNA Ligase IV within an extended flexible NHEJ core complex. The flexible Ku80 C-terminal regions link Ku heterodimers to DNA-PKcs, and APLF interactions stabilize the assembled six-protein complex. Small angle X-ray scattering (SAXS), mutagenesis, in vitro complex assembly The Journal of biological chemistry Medium 27875301
2016 Downregulation of APLF in mouse embryonic fibroblasts promotes reprogramming by augmenting E-cadherin (Cdh1) expression (MET), expediting loss of repressive MacroH2A.1 from the Cdh1 promoter, and enhancing incorporation of active H3me2K4 marks at pluripotency gene promoters. shRNA knockdown, ChIP, immunofluorescence, iPSC reprogramming efficiency assay Journal of cell science Medium 27875275
2017 Crystal structure of the APLF FHA domain bound to phosphorylated XRCC1 peptides reveals a pH-dependent interaction where the phosphoserine/phosphothreonine residues have atypically high pK values. Residues flanking the crystallographic recognition motif enhance binding affinity through non-specific electrostatic interactions, supporting XRCC1-mediated nuclear co-transport of APLF. X-ray crystallography, NMR, fluorescence polarization binding assay Nucleic acids research High 29059378
2018 Crystal structures of the Ku-binding motifs (KBMs) of APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex show that the two motifs bind remote sites on the Ku80 α/β domain. A-KBM and X-KBM independently recruit XRCC4 and XLF, respectively, to laser-irradiated sites via Ku80 binding. Mutation of both KBM binding sites in Ku80 compromises end-joining efficiency and accuracy and increases radiosensitivity. X-ray crystallography, laser microirradiation with live-cell imaging, cellular mutagenesis and end-joining assays, clonogenic survival Nature structural & molecular biology High 30291363
2018 The acidic domain of APLF (APLFAD) is intrinsically disordered and binds both (H3-H4)2 tetramer and H2A-H2B complexes with high affinity. NMR-guided mutational analysis shows that two aromatic side chains in APLFAD anchor to the α1-α2 patches on H2A and H2B, covering most of their DNA-interaction surface, establishing H2A-H2B chaperone activity for APLF. NMR spectroscopy, biochemical binding assay, histone chaperone assay, site-directed mutagenesis Nucleic acids research High 29905837
2022 Crystal structure of the APLFAD-histone octamer complex shows APLFAD tethers histones in their nucleosomal conformation. APLF acidic domain can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes in vitro. Mutations of key aromatic anchor residues in APLFAD impair chaperone activity in vitro and in cells. X-ray crystallography, in vitro nucleosome reconstitution assay, site-directed mutagenesis, cellular functional assay Science advances High 35895815
2022 APLF stabilizes DNA end bridging (synapsis) in NHEJ; together with Ku70-Ku80, it establishes a minimal complex supporting DNA synapsis for several minutes under piconewton forces. The C-terminal acidic region of APLF is critical for DNA end bridging. NIHCOLE lncRNA increases dwell time of synapses formed by Ku70-Ku80 and APLF. Magnetic tweezers single-molecule assay, domain deletion analysis, reconstituted NHEJ complex Cell reports High 36640344
2024 PARP1 activity at stalled replication forks facilitates APLF recruitment to stalled forks via the APLF PBZ domain. APLF is required for FANCD2 recruitment to stalled forks and for protection of nascent DNA from MRE11-dependent degradation. APLF depletion sensitizes cells to cisplatin and impairs interstrand crosslink (ICL) repair. siRNA depletion, DNA fiber assay (fork protection), co-IP, immunofluorescence (FANCD2 and APLF at forks), PARP1 inhibitor treatment, cisplatin sensitivity assay Nucleic acids research Medium 38520407
2024 APLF co-localizes with γ-tubulin at centrosomes in mouse embryonic stem cells and governs centrosome number and integrity via PLK4 phosphorylation. Mouse APLF exhibits kinase activity; residue R37 within the FHA domain is indispensable for this kinase activity and for regulating centrosome number. Immunofluorescence, enzymatic kinase assay, docking studies, site-directed mutagenesis, domain deletion analysis European journal of cell biology Low 38968704
2024 PARP1 facilitates transport of APLF from the cytosol to the nucleus in TNBC cells, and nuclear APLF is associated with EMT-linked metastasis. Inhibition of PARP1 enzymatic activity with olaparib abrogates nuclear APLF expression and reduces EMT gene expression. Subcellular fractionation, NLS-tagged APLF stable expression in MCF7, PARP1 inhibitor treatment, in vitro/in vivo invasion assays Biochimica et biophysica acta. Molecular basis of disease Low 39384105

Source papers

Stage 0 corpus · 26 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2011 PARP-3 and APLF function together to accelerate nonhomologous end-joining. Molecular cell 266 21211721
2011 DNA repair factor APLF is a histone chaperone. Molecular cell 135 21211722
2007 APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Molecular and cellular biology 130 17353262
2012 APLF promotes the assembly and activity of non-homologous end joining protein complexes. The EMBO journal 116 23178593
2018 XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining. Nature structural & molecular biology 92 30291363
2010 Solution structures of the two PBZ domains from human APLF and their interaction with poly(ADP-ribose). Nature structural & molecular biology 88 20098424
2010 Structure and identification of ADP-ribose recognition motifs of APLF and role in the DNA damage response. Proceedings of the National Academy of Sciences of the United States of America 82 20439749
2008 APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells. Molecular and cellular biology 82 18474613
2008 APLF (C2orf13) facilitates nonhomologous end-joining and undergoes ATM-dependent hyperphosphorylation following ionizing radiation. DNA repair 76 18077224
2007 Human Xip1 (C2orf13) is a novel regulator of cellular responses to DNA strand breaks. The Journal of biological chemistry 66 17507382
2016 An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex. The Journal of biological chemistry 61 27875301
2013 The PARP3- and ATM-dependent phosphorylation of APLF facilitates DNA double-strand break repair. Nucleic acids research 54 23449221
2018 DNA repair factor APLF acts as a H2A-H2B histone chaperone through binding its DNA interaction surface. Nucleic acids research 41 29905837
2013 Identification and functional characterization of a Ku-binding motif in aprataxin polynucleotide kinase/phosphatase-like factor (APLF). The Journal of biological chemistry 35 23689425
2019 Rewiring E2F1 with classical NHEJ via APLF suppression promotes bladder cancer invasiveness. Journal of experimental & clinical cancer research : CR 24 31287003
2022 Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF. Science advances 17 35895815
2018 Enhanced expression of histone chaperone APLF associate with breast cancer. Molecular cancer 16 29580241
2022 APLF and long non-coding RNA NIHCOLE promote stable DNA synapsis in non-homologous end joining. Cell reports 14 36640344
2016 Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts. Journal of cell science 11 27875275
2024 APLF facilitates interstrand DNA crosslink repair and replication fork protection to confer cisplatin resistance. Nucleic acids research 10 38520407
2020 End Processing Factor APLF Promotes NHEJ Efficiency and Contributes to TMZ- and Ionizing Radiation-Resistance in Glioblastoma Cells. OncoTargets and therapy 9 33116637
2017 Characterization of the APLF FHA-XRCC1 phosphopeptide interaction and its structural and functional implications. Nucleic acids research 8 29059378
2021 Histone chaperone APLF level dictates the implantation of mouse embryos. Journal of cell science 6 33277378
2025 Aplf/Dna2 variants drive chromosomal fission and accelerate speciation in zokors. Science advances 3 40911670
2024 Kinase activity of histone chaperone APLF maintains steady state of centrosomes in mouse embryonic stem cells. European journal of cell biology 1 38968704
2024 Nuclear localization of APLF facilitates breast cancer metastasis. Biochimica et biophysica acta. Molecular basis of disease 0 39384105

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