Affinage

HPF1

Histone PARylation factor 1 · UniProt Q9NWY4

Length
346 aa
Mass
39.4 kDa
Annotated
2026-04-28
29 papers in source corpus 21 papers cited in narrative 21 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

HPF1 is an accessory factor of PARP1 and PARP2 that remodels the specificity and output of ADP-ribosylation at DNA damage sites, functioning as a central regulator of the DNA damage response. HPF1 transiently engages the PARP1/2 catalytic domain to form a composite active site in which its Glu284 residue serves as the catalytic base, switching ADP-ribosylation from glutamate/aspartate residues to serine residues on histones, PARP1/2 themselves, and other substrates such as HuR and FUS (PMID:28190768, PMID:32028527, PMID:33589610). Operating via a sub-stoichiometric 'hit and run' mechanism, HPF1 initiates serine mono-ADP-ribosylation, limits PARP1 hyper-automodification, modulates PAR chain length, and can convert PARP1 into a hydrolase producing free ADP-ribose; these activities drive chromatin relaxation near double-strand breaks, promoting recruitment of both homologous recombination and non-homologous end joining machineries, while HPF1-dependent serine ADP-ribosylation is largely dispensable for single-strand break repair (PMID:34795260, PMID:33683197, PMID:37106138, PMID:39162207). HPF1 also influences PARP inhibitor pharmacology by altering drug dissociation kinetics from the PARP1–HPF1 complex and supports backup Okazaki fragment ligation and base excision repair through PARP1/2-dependent histone modification (PMID:37531469, PMID:33872376, PMID:40076422).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2016 High

    The identity and basic function of HPF1 as a PARP1-interacting factor were unknown; its discovery established that C4orf27/HPF1 binds PARP1, is recruited to DNA lesions, promotes histone ADP-ribosylation in trans, and restrains PARP1 automodification.

    Evidence Co-immunoprecipitation, live-cell recruitment assays, in vitro ADP-ribosylation, and HPF1 knockout cells

    PMID:27067600

    Open questions at the time
    • Amino acid specificity of HPF1-directed modification unknown
    • Structural basis of HPF1–PARP1 interaction unresolved
    • In vivo DNA repair pathway consequences not defined
  2. 2017 High

    The amino acid specificity question was resolved: HPF1 is necessary and sufficient to redirect PARP1/2 modification from aspartate/glutamate to serine, establishing serine ADP-ribosylation as the dominant DNA-damage-induced modification.

    Evidence Quantitative ADPr site mapping proteomics in HPF1-KO cells and in vitro reconstitution with recombinant HPF1

    PMID:28190768

    Open questions at the time
    • Structural mechanism of specificity switch unknown
    • Identity of catalytic residue(s) on HPF1 not established
  3. 2020 High

    Structural studies revealed how HPF1 achieves serine specificity: it forms a composite active site with the PARP1/2 catalytic domain, contributing Glu284 as the catalytic base, with DNA damage and NAD⁺ occupancy allosterically promoting the assembly; cryo-EM further showed PARP2–HPF1 bridges nucleosomes across a DSB.

    Evidence Co-crystal structure of HPF1–PARP2-CAT (X-ray), cryo-EM of PARP2–HPF1–nucleosome complexes, NMR, mutagenesis, and chemoenzymatic reconstitution

    PMID:32028527 PMID:32939087 PMID:33141820 PMID:33186521

    Open questions at the time
    • Full-length PARP1–HPF1 structural dynamics on DNA not resolved
    • Role of individual HPF1 residues beyond Glu284 incompletely mapped
    • How HPF1 binding is regulated in the cell unknown
  4. 2021 High

    Mechanistic studies established that HPF1 operates sub-stoichiometrically via rapid association/dissociation ('hit and run'), controls PAR chain length, converts PARP1 into a hydrolase producing free ADP-ribose, drives chromatin unfolding at DSBs to promote HR and NHEJ factor recruitment, and is required for backup Okazaki fragment ligation via LIG3–XRCC1.

    Evidence Biochemical kinetics, nucleosome PARylation product analysis, live-cell chromatin compaction assays, HPF1-KO phenotyping, and Xenopus egg extract immunodepletion

    PMID:33683197 PMID:33872376 PMID:34795260 PMID:37106138

    Open questions at the time
    • Whether hydrolase switching occurs in vivo not demonstrated
    • Quantitative cellular stoichiometry of HPF1 relative to PARP1/2 foci not measured directly
    • Relative contribution of chromatin relaxation vs. direct signaling to repair factor recruitment unclear
  5. 2021 High

    High-resolution crystal structures of the human HPF1–PARP1-CAT complex confirmed the Arg239–Glu284/Asp286 salt bridge as critical for positioning the catalytic base and maintaining HPF1 conformation that limits PARP1 automodification.

    Evidence X-ray crystallography of HPF1–PARP1-CAT (1.98 Å), HPF1 alone structures, mutagenesis, and binding assays

    PMID:33589610

    Open questions at the time
    • Dynamics of the HPF1–PARP1 interface at the single-molecule level not captured
    • How the composite active site accommodates different histone substrates not structurally defined
  6. 2023 High

    HPF1's role in PARP inhibitor pharmacology was quantified: slow koff of inhibitors from the PARP1–HPF1 complex, rather than from PARP1 alone, best predicts cellular potency, redefining the pharmacologically relevant target complex.

    Evidence Binding kinetics (kon, koff, KD) for eight PARP inhibitors against PARP1 and PARP1–HPF1, correlated with cellular potency

    PMID:37531469

    Open questions at the time
    • Whether HPF1 levels vary across tumor types and affect PARPi sensitivity in patients unknown
    • Structural basis of HPF1-induced changes in inhibitor binding not resolved
  7. 2024 High

    The boundaries of HPF1 function were defined: HPF1 loss reduces serine mono-ADP-ribosylation at SSBs but does not substantially impair PAR signaling or XRCC1 recruitment, establishing that HPF1-dependent serine modification is largely dispensable for single-strand break repair.

    Evidence HPF1-KO cells, SSB repair kinetics, PAR and XRCC1 quantification at SSB sites, genotoxin sensitivity assays

    PMID:39162207

    Open questions at the time
    • Whether HPF1-independent repair at SSBs relies on glutamate/aspartate PARylation not formally tested
    • Role of HPF1 in replication-associated SSBs not addressed
  8. 2025 Medium

    HPF1 was shown to modulate BER efficiency: it enhances total PARylation by PARP1/2 on nucleosomes in ways that positively affect both short-patch and long-patch BER DNA synthesis by pol β.

    Evidence In vitro reconstituted BER assays with nucleosomes and varying HPF1/PARP1/PARP2

    PMID:40076422

    Open questions at the time
    • Cellular validation of HPF1 role in BER not performed
    • Whether HPF1-dependent serine vs. glutamate modification differentially affects BER unknown
  9. 2026 High

    Structural and biophysical studies of full-length PARP1 on SSB DNA with HPF1 revealed that the catalytic region remains highly mobile, suggesting a tethered, constitutively active catalytic domain that modifies molecules in a radius around the break; separately, HPF1 was shown to stimulate hetero-PARylation of FUS, impairing DNA-rich FUS compartment assembly.

    Evidence Cryo-EM of full-length PARP1–HPF1 on nicked DNA plus SAXS and single-molecule dynamics; AFM and in vitro reconstitution of FUS compartments with PARP1/PARP2/HPF1

    PMID:41698892 PMID:41773021

    Open questions at the time
    • Functional consequences of catalytic domain mobility for substrate selection in cells not tested
    • Physiological relevance of FUS PARylation by HPF1–PARP1/2 unclear
    • Full atomic model of PARP1–HPF1 on damaged chromatin still lacking

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key open questions include: the in vivo regulation of HPF1 expression and turnover, the structural basis of HPF1's effects on PARP inhibitor binding, whether HPF1-dependent serine ADP-ribosylation has non-chromatin functions (e.g., ribosomal protein modification, phase separation regulation), and whether HPF1 levels influence PARP inhibitor sensitivity in clinical settings.
  • No in vivo regulation or post-translational modification of HPF1 characterized
  • No disease-associated mutations in HPF1 reported
  • Ribosomal substrate specificity (60S preference) awaits cellular validation

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 6 GO:0140096 catalytic activity, acting on a protein 4
Localization
GO:0005694 chromosome 5 GO:0005634 nucleus 2
Pathway
R-HSA-73894 DNA Repair 5 R-HSA-4839726 Chromatin organization 3 R-HSA-69306 DNA Replication 1
Partners
ELAVL1FUSLIG3PARP1PARP2XRCC1
Complex memberships
PARP1–HPF1 composite active-site complexPARP2–HPF1 composite active-site complex

Evidence

Reading pass · 21 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2016 HPF1 (C4orf27) forms a robust protein complex with PARP1 in cells, is recruited to DNA lesions in a PARP1-dependent but catalytic-activity-independent manner, promotes PARP1-dependent in trans ADP-ribosylation of histones, and limits DNA damage-induced hyper-automodification of PARP1. Co-immunoprecipitation, live-cell recruitment assays, in vitro ADP-ribosylation assays, loss-of-function (HPF1 knockout cells) Molecular cell High 27067600
2017 HPF1 is necessary and sufficient for serine-specific ADP-ribosylation by PARP-1 and PARP-2; serine ADPr does not occur in HPF1-null cells, and adding HPF1 to in vitro PARP-1/PARP-2 reactions redirects modification from aspartate/glutamate to serine residues on histones and PARP-1 itself. Quantitative proteomics (ADPr site mapping), in vitro reconstitution with recombinant HPF1, HPF1 knockout cell lines Molecular cell High 28190768
2020 HPF1 forms a composite active site with the catalytic domain of PARP1 or PARP2, contributing the catalytic base Glu284 to switch amino acid specificity from aspartate/glutamate to serine; this assembly is allosterically enhanced by DNA damage and NAD⁺-binding site occupancy. Co-crystal structure of HPF1 bound to PARP2 catalytic domain (X-ray crystallography), NMR, biochemical mutagenesis, cellular ADP-ribosylation assays Nature High 32028527
2020 PARP2-HPF1 bridges two nucleosomes across a DNA double-strand break, with broken DNA aligned for ligation; this bridging induces structural changes in PARP2 that signal DNA-break recognition to the catalytic domain, licensing HPF1 binding and PARP2 activation. Cryo-EM structure of human PARP2-HPF1 bound to a nucleosome pair, biochemical activity assays Nature High 32939087
2020 An HPF1/PARP1-based chemoenzymatic approach using phospho-guided serine ADP-ribosylation enables scalable preparation of site-specifically ADP-ribosylated peptides, confirming that the HPF1/PARP1 writer complex installs serine-linked mono-ADPr and that PARG and ARH3 are the primary erasers. Chemoenzymatic in vitro reconstitution, mono-ADPr proteomics, phage display antibody selection Cell High 33186521
2020 PARP2 bridges DNA double-strand break ends in the context of nucleosomes with short linker DNA, and this nucleosome-bridged conformation provides a binding platform for HPF1 that is required for enzymatic activation of the PARP2-HPF1 complex. Cryo-EM structure of PARP2 bridging two nucleosomes, biochemical activity assays of HPF1·PARP2·nucleosome complex PloS one High 33141820
2021 Crystal structures of human HPF1/PARP1-CAT complex confirm that HPF1 Arg239 salt-bridges to Glu284/Asp286, positioning Glu284 as the catalytic base for serine ADP-ribosylation, maintaining HPF1 conformation to limit PARP1 automodification, and facilitating HPF1/PARP1 binding. X-ray crystal structures of HPF1/PARP1-CAT complex (1.98 Å), HPF1 alone (1.71 Å mouse, 1.57 Å human), mutagenesis of key residues, quantitative binding assays Nature communications High 33589610
2021 HPF1 operates via a 'hit and run' mechanism: it rapidly associates and dissociates from multiple PARP1 molecules at sub-stoichiometric ratios matching cellular abundances, initiating serine ADP-ribosylation before glutamate/aspartate modification initiates, and then dissociates to allow PAR chain elongation without interference. Biochemical kinetic assays, structural analysis of HPF1/PARP1 on DNA break, PARP1 DNA retention assays Nature communications High 34795260
2021 HPF1 provides the catalytic base Glu284 to redirect PARP1 PARylation so that histones in nucleosomes become the primary PAR recipients; unexpectedly, HPF1 also partitions most reaction product to free ADP-ribose (ADPR), switching PARP1 from a polymerase to a hydrolase and producing shorter PAR chains. In vitro PARP1 activity assays with nucleosomes, biochemical product analysis (PAR chain length, free ADPR quantification), mutagenesis eLife High 33683197
2021 HPF1 controls both the number and length of ADP-ribose chains on histones near DNA breaks, and HPF1-dependent histone ADP-ribosylation triggers rapid chromatin unfolding that promotes assembly of both homologous recombination and non-homologous end joining repair machineries. Live-cell imaging (chromatin compaction assays), HPF1 knockout cells, PARP1 inhibition, quantitative analysis of HR and NHEJ factor recruitment Nature structural & molecular biology High 37106138
2021 HPF1 can both stimulate and limit PARP1/PARP2 activity in a concentration- and NAD⁺-dependent manner: at defined HPF1/NAD⁺ concentrations it stimulates autoPARylation of PARP1/PARP2 and heteroPARylation of histones in nucleosomes without triggering hydrolytic NAD⁺ consumption; PARP2 is more efficiently stimulated by HPF1 than PARP1 in autoPARylation. In vitro PARylation assays with purified components, nucleosome substrates, varying HPF1 and NAD⁺ concentrations Communications biology Medium 34732825
2021 HPF1-dependent PARP1 activation promotes ADP-ribosylation of histone H3 and is essential for LIG3-XRCC1 recruitment to chromatin as a backup Okazaki fragment ligation pathway when LIG1 is absent. Xenopus egg extract cell-free system, immunodepletion of PARP1 and HPF1, chromatin recruitment assays, Okazaki fragment ligation assays Nucleic acids research High 33872376
2022 PARP2-HPF1-dependent histone PARylation is specifically stimulated by a gapped nucleosome containing a 5'-dRP group (BER intermediate), and this HPF1-induced stimulation of histone heteromodification is a peculiar feature of PARP2, suggesting a specific regulatory role of PARP2 in chromatin-context DNA repair. In vitro PARylation assays with defined nucleosome substrates containing specific DNA lesions, affinity binding measurements DNA repair Medium 36356486
2023 Slow dissociation (koff) of PARP inhibitors from the PARP1-HPF1 complex, rather than from PARP1 alone, is the parameter that best correlates with inhibitor potency in cells; HPF1 increases the affinity of certain PARPi (e.g., fluzoparib, olaparib) for PARP1. Binding kinetics (kon, koff, KD) measurements for eight PARP inhibitors against PARP1 and PARP1-HPF1 complex, correlation with cellular potency data Biochemistry High 37531469
2023 HPF1 promotes PARP1-mediated poly-ADP-ribosylation of HuR, and this modification regulates the interaction of HuR with p16 and p21 mRNAs, thereby controlling their half-lives and modulating tendon stem/progenitor cell senescence. Co-immunoprecipitation (HPF1-PARP1 interaction), RNP-IP (HuR-mRNA binding), IP for PARylation of HuR, mRNA half-life assays, siRNA knockdown/overexpression Genes & genomics Medium 37713069
2024 HPF1 deficiency reduces serine mono-ADP-ribosylation at DNA single-strand break sites but does not substantially impair poly-ADP-ribosylation or XRCC1 recruitment, demonstrating that HPF1-dependent serine ADP-ribosylation is largely dispensable for PARP1-dependent single-strand break repair. HPF1 knockout cells, SSBR kinetics assays, quantification of poly-ADP-ribosylation and XRCC1 recruitment at SSB sites, genotoxin sensitivity assays Nucleic acids research High 39162207
2025 HPF1 modulates total poly(ADP-ribosyl)ation by PARP1 and especially PARP2, and this regulation positively affects short-patch BER DNA synthesis by DNA polymerase β and long-patch BER efficiency in nucleosome context. In vitro reconstituted BER assays with nucleosomes, quantification of DNA synthesis by pol β under varying HPF1/PARP1/PARP2 conditions International journal of molecular sciences Medium 40076422
2026 HPF1 stimulates hetero-PARylation of FUS (an RNA-binding protein) by PARP1 and PARP2; HPF1-dependent intensive PARylation of FUS impairs assembly of DNA-rich compartments, while excess HPF1 reduces PARP1 (but not PARP2) activity and the size of PAR-dependent FUS compartments. Atomic force microscopy, biochemical PARylation assays, in vitro reconstitution of FUS compartment formation with PARP1/PARP2/HPF1 Nucleic acids research Medium 41773021
2026 Full-length PARP1 assembled on a DNA single-strand break with HPF1 remains structurally dynamic by cryo-EM, with the catalytic region displaying high mobility relative to DNA-damage-engaging domains, suggesting the organized multi-domain structure releases a tethered, constitutively active catalytic region to modify molecules in a radius around the break. Single-particle cryo-EM of full-length PARP1 on nicked DNA with HPF1 and Timeless fragment, single-molecule DNA dynamics, SAXS Nature communications High 41698892
2025 HPF1 switches ADP-ribosylation of free ribosomal large subunit proteins (60S) preferentially to serine/tyrosine residues, with RPL4, RPL6, and RPL13A/RPL15 as major targets; HPF1-enhanced PARylation is specific to 60S proteins and does not significantly affect 40S ribosomal proteins. In vitro ADP-ribosylation assays with radioactively labeled NAD⁺, ribosomal subunit fractionation, site identification bioRxivpreprint Low bio_10.1101_2025.09.15.676193
2025 In the presence of HPF1, PARP2 serine 8 (present in both human PARP2 isoforms) and serine 73 are the predominant automodification sites; ADP-ribosylation of serine 8 drives PARP2 release from DNA damage sites. Site-directed mutagenesis, gel-based PARylation assays, fluorescence polarization assay for PARP2-DNA release bioRxivpreprint Medium bio_10.1101_2025.07.01.662498

Source papers

Stage 0 corpus · 29 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2017 Serine ADP-Ribosylation Depends on HPF1. Molecular cell 294 28190768
2016 HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity. Molecular cell 258 27067600
2020 HPF1 completes the PARP active site for DNA damage-induced ADP-ribosylation. Nature 221 32028527
2020 Bridging of DNA breaks activates PARP2-HPF1 to modify chromatin. Nature 112 32939087
2020 An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation. Cell 89 33186521
2021 HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones. Nature communications 70 33589610
2021 HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications. Nature communications 60 34795260
2021 HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase. eLife 55 33683197
2023 HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage. Nature structural & molecular biology 54 37106138
2008 Radical-scavenging abilities and antioxidant properties of theaflavins and their gallate esters in H2O2-mediated oxidative damage system in the HPF-1 cells. Toxicology in vitro : an international journal published in association with BIBRA 52 18502093
2021 HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation. Nucleic acids research 44 33872376
2020 Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1. PloS one 37 33141820
2021 Dual function of HPF1 in the modulation of PARP1 and PARP2 activities. Communications biology 27 34732825
2020 Intragenic repeat expansion in the cell wall protein gene HPF1 controls yeast chronological aging. Genome research 25 32277013
2024 Dispensability of HPF1 for cellular removal of DNA single-strand breaks. Nucleic acids research 15 39162207
2023 Slow Dissociation from the PARP1-HPF1 Complex Drives Inhibitor Potency. Biochemistry 12 37531469
2017 SERious Surprises for ADP-Ribosylation Specificity: HPF1 Switches PARP1 Specificity to Ser Residues. Molecular cell 10 28257697
2022 The HPF1-dependent histone PARylation catalyzed by PARP2 is specifically stimulated by an incised AP site-containing BER DNA intermediate. DNA repair 9 36356486
2024 High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair. Scientific reports 7 38365924
2023 HTS discovery of PARP1-HPF1 complex inhibitors in cancer. SLAS discovery : advancing life sciences R & D 5 37844763
2023 HPF1 regulates tendon stem/progenitor cell senescence and tendon repair via PARP1-mediated poly-ADP ribosylation of HuR. Genes & genomics 4 37713069
2018 Analysis of hpf1 expression and function in early embryonic development of zebrafish. Development genes and evolution 4 29549427
2023 [Poly(ADP-Ribose) Polymerases 1 and 2: Classical Functions and Interaction with New Histone Poly(ADP-Ribosyl)ation Factor HPF1]. Molekuliarnaia biologiia 3 37000654
2023 ANP32B promotes colorectal cancer cell progression and reduces cell sensitivity to PRAP1 inhibitor through up-regulating HPF1. Heliyon 3 38192816
2025 HPF1 Regulates Pol β Efficiency in Nucleosomes via the Modulation of Total Poly(ADP-Ribose) Synthesis. International journal of molecular sciences 1 40076422
2023 Germline HPF1 retrogene insertion in RB1 gene involved in cancer predisposition. Journal of medical genetics 1 37541786
2026 PARP1-HPF1 structure and dynamics on nicked DNA suggest a mechanism for acute and localized ADP-ribosylation. Nature communications 0 41698892
2026 HPF1 regulates the formation of FUS-dependent compartments by PARP1 and PARP2 activation on damaged DNA. Nucleic acids research 0 41773021
2026 The Structure of the Nucleosomal DNA Repair Intermediate Affects the HPF1-Independent Automodification Activity of PARP2. Biochemistry. Biokhimiia 0 41936561