Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1,2,3,4,5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6,7,8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2–HPF1 bound to a nucleosome. This showed that PARP2–HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12,13,14,15,16.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Structural dynamics of DNA strand break sensing by PARP-1 at a single-molecule level
Nature Communications Open Access 02 November 2022
The synthetic lethality of targeting cell cycle checkpoints and PARPs in cancer treatment
Journal of Hematology & Oncology Open Access 17 October 2022
G4-quadruplex-binding proteins: review and insights into selectivity
Biophysical Reviews Open Access 20 April 2022
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 per month
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
EM densities have been deposited in the Electron Microscopy Data Bank under accession codes EMD-21980, EMD-21971, EMD-21970, EMD-21978, EMD-21979, EMD-21981 and EMD-21982. The coordinates of EM-based models have been deposited in the Protein Data Bank under accession codes PDB 6X0N, 6X0L, 6X0M, 6WZ9 and 6WZ5. The RAW data are provided as a Supplementary Fig. All other data are available from the corresponding author upon reasonable request.
Liu, C., Vyas, A., Kassab, M. A., Singh, A. K. & Yu, X. The role of poly ADP-ribosylation in the first wave of DNA damage response. Nucleic Acids Res. 45, 8129–8141 (2017).
Ray Chaudhuri, A. & Nussenzweig, A. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat. Rev. Mol. Cell Biol. 18, 610–621 (2017).
Gupte, R., Liu, Z. & Kraus, W. L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes. Genes Dev. 31, 101–126 (2017).
Caldecott, K. W. Protein ADP-ribosylation and the cellular response to DNA strand breaks. DNA Repair (Amst.) 19, 108–113 (2014).
Pascal, J. M. The comings and goings of PARP-1 in response to DNA damage. DNA Repair (Amst.) 71, 177–182 (2018).
Leidecker, O. et al. Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat. Chem. Biol. 12, 998–1000 (2016).
Bonfiglio, J. J. et al. Serine ADP-ribosylation depends on HPF1. Mol. Cell 65, 932–940.e6 (2017).
Larsen, S. C., Hendriks, I. A., Lyon, D., Jensen, L. J. & Nielsen, M. L. Systems-wide analysis of serine ADP-ribosylation reveals widespread occurrence and site-specific overlap with phosphorylation. Cell Rep. 24, 2493–2505.e4 (2018).
Gibbs-Seymour, I., Fontana, P., Rack, J. G. M. & Ahel, I. HPF1/C4orf27 is a PARP-1-interacting protein that regulates PARP-1 ADP-ribosylation activity. Mol. Cell 62, 432–442 (2016).
Palazzo, L. et al. Serine is the major residue for ADP-ribosylation upon DNA damage. eLife 7, e34334 (2018).
Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).
Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).
Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).
Hou, W.-H., Chen, S.-H. & Yu, X. Poly-ADP ribosylation in DNA damage response and cancer therapy. Mutat. Res. 780, 82–91 (2019).
Lord, C. J. & Ashworth, A. PARP inhibitors: synthetic lethality in the clinic. Science 355, 1152–1158 (2017).
Murai, J. et al. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 72, 5588–5599 (2012).
Bilokapic, S., Strauss, M. & Halic, M. Histone octamer rearranges to adapt to DNA unwrapping. Nat. Struct. Mol. Biol. 25, 101–108 (2018).
Suskiewicz, M. J. et al. HPF1 completes the PARP active site for DNA damage-induced ADP-ribosylation. Nature 579, 598–602 (2020).
Obaji, E., Haikarainen, T. & Lehtiö, L. Structural basis for DNA break recognition by ARTD2/PARP2. Nucleic Acids Res. 46, 12154–12165 (2018).
Riccio, A. A., Cingolani, G. & Pascal, J. M. PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage. Nucleic Acids Res. 44, 1691–1702 (2016).
Langelier, M.-F., Riccio, A. A. & Pascal, J. M. PARP-2 and PARP-3 are selectively activated by 5′ phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1. Nucleic Acids Res. 42, 7762–7775 (2014).
Langelier, M.-F., Planck, J. L., Roy, S. & Pascal, J. M. Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1. Science 336, 728–732 (2012).
Dawicki-McKenna, J. M. et al. PARP-1 activation requires local unfolding of an autoinhibitory domain. Mol. Cell 60, 755–768 (2015).
Thorsell, A.-G. et al. Structural basis for potency and promiscuity in poly(ADP-ribose) polymerase (PARP) and tankyrase inhibitors. J. Med. Chem. 60, 1262–1271 (2017).
Langelier, M.-F., Zandarashvili, L., Aguiar, P. M., Black, B. E. & Pascal, J. M. NAD+ analog reveals PARP-1 substrate-blocking mechanism and allosteric communication from catalytic center to DNA-binding domains. Nat. Commun. 9, 844 (2018).
Kim, M. Y., Mauro, S., Gévry, N., Lis, J. T. & Kraus, W. L. NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1. Cell 119, 803–814 (2004).
Muthurajan, U. M. et al. Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone. Proc. Natl Acad. Sci. USA 111, 12752–12757 (2014).
Yap, T. A., Plummer, R., Azad, N. S. & Helleday, T. The DNA damaging revolution: PARP inhibitors and beyond. Am. Soc. Clin. Oncol. Educ. Book 39, 185–195 (2019).
Pettitt, S. J. et al. Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance. Nat. Commun. 9, 1849 (2018).
Zandarashvili, L. et al. Structural basis for allosteric PARP-1 retention on DNA breaks. Science 368, eaax6367 (2020).
Luger, K., Rechsteiner, T. J., Flaus, A. J., Waye, M. M. & Richmond, T. J. Characterization of nucleosome core particles containing histone proteins made in bacteria. J. Mol. Biol. 272, 301–311 (1997).
Bilokapic, S., Strauss, M. & Halic, M. Cryo-EM of nucleosome core particle interactions in trans. Sci. Rep. 8, 7046 (2018).
Bilokapic, S., Strauss, M. & Halic, M. Structural rearrangements of the histone octamer translocate DNA. Nat. Commun. 9, 1330 (2018).
Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998).
Bilokapic, S. & Halic, M. Nucleosome and ubiquitin position Set2 to methylate H3K36. Nat. Commun. 10, 3795 (2019).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015).
Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
We thank A. Myasnikov and L. Tang from the Cryo EM facility at St Jude Children's Research Hospital for support with the data collection. M.J.S. is supported by EMBO Long-term Fellowship ALTF 879-2017. Work in I.A.’s laboratory is funded by the Wellcome Trust (grants 101794 and 210634), BBSRC (BB/R007195/1) and Cancer Research UK (C35050/A22284). Work in M.H.’s laboratory is funded by St Jude Children's Research Hospital, the American Lebanese Syrian Associated Charities and US NIH award 1R01GM135599-01.
The authors declare no competing interests.
Peer review information Nature thanks Ivan Dikic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Assembly and cryo-EM of PARP2–HPF1 bound to mononucleosomes.
a, b, SDS–PAGE (a) and native gel (b) showing the PARP2–HPF1–nucleosome complex assembly for cryo-EM. Note the shift in the PARP2–nucleosome complex migration upon binding of HPF1 in b. c, SDS–PAGE and immunoblotting showing PARP2 PARylation of nucleosomes. HPF1 is required for H3 PARylation. d, Representative cryo-EM micrograph collected with Titan Krios electron microscope at 300 keV. Bridging of two nucleosomes by PARP2–HPF1 is clearly visible in the raw data. Complex particles in multiple orientations are visible. e, Representative 2D class averages showing two nucleosomes bridged by PARP2–HPF1. Two nucleosomes are positioned in an almost perpendicular orientation. PARP2–HPF1 density between two nucleosomes is clearly visible. Many details in nucleosomes are visible in 2D class averages. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 Classification of the PARP2–HPF1–nucleosome complex.
a, Initial map generated from the entire data set, comprising 934,000 particles. The data set was further extensively classified. The regions used for focused classifications and refinements in b–g are colour coded and labelled. b, Cryo-EM map of nucleosome 2, refined to 2.2 Å, is shown on the left. Fourier shell correlation (FSC) curve showing the resolution of the map (middle). The map is coloured by local resolution. The model of the NCP (PDB 6FQ5) was refined into the cryo-EM map. The representative region showing map quality and fit of the model is on the right. c, Angular distribution for nucleosome 2. d, Directional FSC plot showing uniform resolution in all directions. e, Cryo-EM map of nucleosome 1, refined to 2.8 Å, is shown on the left. Fourier shell correlation (FSC) curve showing the resolution of the map (middle). The map is coloured by local resolution. The model of the NCP (PDB 6FQ5) was refined into the cryo-EM map. The representative region showing map quality and fit of the model is on the right. f, Focused classification and refinements with a focus on the connection of the PARP2 WGR domains with nucleosome 2 (see b). Cryo-EM map of this region was refined to 4.1 Å (left). FSC curve showing the resolution of the map is in the middle. The model of the PARP2 WGR domains bound to DNA (PDB 6F5B) was refined into the cryo-EM map. The representative region showing map quality and fit of the model is on the right. g, Focused classification and refinements with focus on the connection of the PARP2 WGR domains with nucleosome 1 (e). Cryo-EM map of this region was refined to 5.7 Å (left). FSC curve showing the resolution of the map is in the middle. The model of the PARP2 WGR domains bound to DNA (PDB 6F5B) was refined into the cryo-EM map. The representative region showing map quality and fit of the model is on the right.
Extended Data Fig. 3 Focused classification, refinement and model building: focus on the PARP2–HPF1 complex.
a, The overall data set, comprising 934,000 particles, was extensively classified with focus on PARP2–HPF1 complex found in between the two nucleosomes. b, Cryo-EM map of PARP2–HPF1 bound to the nucleosome in the activated conformation. Cryo-EM map of this conformation was refined to 4.2 Å. FSC curve showing the resolution of the map is below. c, Cryo-EM map of PARP2–HPF1 bound to the nucleosome in open state 1. Cryo-EM map of this conformation was refined to 6.7 Å. FSC curve showing the resolution of the map is below. The map is coloured by local resolution. d, Cryo-EM map of PARP2–HPF1 bound to the nucleosome in open state 2. Cryo-EM map of this conformation was refined to 6.3 Å. FSC curve showing the resolution of the map is shown below. The map is coloured by local resolution. e, The PARP2–HPF1 subset comprising 32,000 particles (left) was further classified and refined. Protruding DNA was eliminated from refinements to improve the resolution. Final map (middle) was refined to 3.9 Å. FSC curve is shown on the right. The map is coloured by local resolution. f, Angular distribution for PARP2–HPF1. g, Directional FSC plot showing reasonably uniform resolution in all directions. h, The models of the PARP2 WGR domain (PDB 6F5B), PARP2 catalytic domain (CAT) with HD domain (PDB 4TVJ) and PARP2 catalytic domain without HD domain in complex with HPF1 (PDB 6TX3) were refined into the cryo-EM map. The representative regions showing map quality and fit of the model are shown. Side chains are visible in most regions of the map.
Extended Data Fig. 4 PARP2 interaction with nucleosomes.
a, The PARP2–HPF1 subset comprising 140,000 particles (left) was further classified and refined. The final map containing two PARP2–HPF1 complexes (middle) was refined to 6.3 Å. FSC curve is shown on the right. The map is coloured by local resolution. b, Model of activated PARP2–HPF1 (Fig. 1a) was rigid body fitted into cryo-EM map of two PARP2–HPF1 complexes bridging two mononucleosomes. c, Schematic representation of PARP2 and HPF1 organization is shown on the left. On the right, PARP2–HPF1 model is coloured by domains. d, Map quality and fit of the model are shown for the region shown in Fig. 1d. e, Alignment of PARP2–HPF1 bridging two nucleosomes with 5′-phosphate (5′ P) DNA (PARP2 WGR domains are shown in violet and pink, DNA in grey) with the X-ray structure of isolated PARP2 WGR domains bound to double-strand DNA with 5′ P (yellow; PDB 6F5B). f, Alignment of PARP2–HPF1 bridging two nucleosomes with 5′ P DNA, with the X-ray structure of isolated PARP2 WGR domains bound to double-strand DNA with 5′ P. The model is coloured by r.m.s. deviation (r.m.s.d.). g, Map quality and fit of the model are shown for the regions shown in Fig. 1e.
Extended Data Fig. 5 Interaction of HPF1 with the nucleosome stabilizes the PARP2–HPF1–nucleosome complex.
a, Native gel showing the PARP2–HPF1–nucleosome complex assembly with equimolar amounts of free DNA and nucleosomes. Nucleosomal and free DNA are labelled with Alexa 488. PARP2–HPF1 binds nucleosomes with higher affinity than free DNA. b, EMSA analysis of the assembly of PARP2–nucleosome and PARP2–HPF1–nucleosome complexes. HPF1 contributes to stability of the complex. Native gel is stained with SYBR Gold. c, SDS–PAGE showing quality of wild-type and mutant HPF1 proteins. d, EMSA analysis of PARP2–HPF1–nucleosome complex assembly with wild-type and mutant HPF1. Mutations in loops that interact with nucleosomal DNA destabilize the complex. Native gel is stained with SYBR Gold. e, One PARP2–HPF1 in the map with two PARP2–HPF1 complexes shows flexibility in the N-terminal region of HPF1. Note an additional density spanning from HPF1 to the double-strand DNA break site. This density could be generated by missing HPF1 helices, HPF1 and the PARP2 N-terminal tail or the H3 tail. f, Superposition of the PARP2 catalytic domains from the PARP2–HPF1 crystal structure (grey; PDB 6TX3) and the PARP2–HPF1–nucleosome cryo-EM model (violet and magenta). HPF1 is slightly rearranged in the cryo-EM structure as compared to the X-ray structure. g, Superposition of the PARP2 catalytic domains from the PARP2–HPF1 crystal structure (grey; PDB 6TX3) and the PARP2–HPF1–nucleosome cryo-EM model. The model is coloured by r.m.s.d. One representative experiment of at least 3 independent experiments is shown for all biochemical data. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6 Bridging of two nucleosomes is required for PARP2 activation.
a, PARP2 can bind double-stranded DNA with 5′-biotin on both ends but cannot bridge that DNA. Complex formation was followed on the native gel through staining with SYBR Gold and anti-PARP2 western blot analysis. b, Native gel showing PARP2 binding to 5′-P and 5′-OH hairpin DNA. This generated two distinct complexes separated on a native gel: PARP2 bound to one DNA and PARP2 bridging two DNAs. PARP2 efficiently bridges hairpin DNA with 5′ P, and only weakly DNA with 5′ OH. Lanes used for PARylation reaction in c are marked *. c, Lanes with equal amounts of PARP2 bridging two DNA from b (marked *) were incubated with NAD+ for 7 min to perform in-gel PARylation assay. 2.5× more PARP2 was required to obtain same amount of the bridged complex with 5′ OH DNA. PARP2 is activated to the same extent by the bridged 5′ P and 5′ OH DNA. Only PARP2 bridging two DNAs shows strong ADP-ribosylation activity. d, SDS–PAGE showing PARP2 auto-PARylation activity. PARP2 was incubated with 5′ P and 5′ OH hairpin DNA and NAD+ in solution under conditions from b, labelled with + in b. PARP2 is activated more strongly by 5′ P hairpin DNA, which forms more stable bridged complex. e, SDS–PAGE showing quality of wild-type and two mutant PARP2 proteins. f, SDS–PAGE showing PARP2 auto-PARylation activity. Wild-type and mutant PARP2 were incubated with NAD+ with or without DNA. Note increased DNA-independent activity and reduction in DNA-dependent activity for PARP2 mutants. g, Native gel and anti-H3 western blot showing PARP2–nucleosome and PARP2–HPF1–nucleosome complex assembly with wild-type and mutant PARP2. The mutation of PARP2 R140, which bridges two nucleosomes, abolishes complex formation, whereas the mutation of PARP2 V141 reduces complex stability. Note that complexes with HPF1 show higher stability. One representative experiment of at least 3 independent experiments is shown for all biochemical data. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 Bridging of two nucleosomes induces conformational changes in PARP2.
a, Alignment of WGR domains of PARP2 cryo-EM model (violet) and PARP1 DNA bound X-ray structure (grey; PDB 4DQY). Note conformational changes in the CAT_HD domain, especially in helices αA, αB, αF and αG. Red box shows the location of the zoom-in view in the overall structure b, Alignment of WGR domains of PARP2 cryo-EM model (violet) and PARP1 DNA bound X-ray structure (grey; PDB 4DQY). The model is coloured by r.m.s.d. Note conformational changes in the CAT_HD domain, especially in helices αA, αB, αF and αG. c, Close-up view of alignment of catalytic ART domains of PARP2 cryo-EM model (violet) and PARP2 catalytic domain X-ray structure (grey; PDB 4TVJ). Note conformational changes in αB and in the hydrophobic and HD loops. d, Close-up view of the PARP2 WGR signalling loop interaction with the hydrophobic loop and the HD loop in the HD subdomain. Map quality and fit of the model are shown for the region shown in Fig. 2d. The side chains building the hydrophobic pocket are resolved in the map. e, Close-up view of PARP2 hydrophobic pocket as in Fig. 2e. The map and the fit of the model are shown for the hydrophobic pocket. Side chains are resolved. The residues building the hydrophobic pocket are labelled. f, Point mutations in PARP1 and PARP2 showing increase in DNA-independent activity are labelled as red sticks. g, The map and the fit of the model for the NAD+-binding site.
Extended Data Fig. 8 Model for PARP2–HPF1 in open state 1.
a, Model of activated PARP2–HPF1 bound to nucleosome (Fig. 1a) was rigid body fitted into the cryo-EM map of the PARP2–HPF1 open state 1 (Extended Data Fig. 3c). PARP2 and HPF1 secondary structure elements are resolved in the cryo-EM map, and the model can be fitted as rigid body. Model is shown in green and the cryo-EM map in transparent green. b, Model of PARP2–HPF1 from a is shown fitted into the cryo-EM map. Several PARP2 helices are flexible in this conformation and are not visible in the cryo-EM map. PARP2 helix αE is partially visible at this contour level, and present at lower contour. c, NAD+ is shown with the cryo-EM map to depict accessibility to the NAD+-binding site. Flexibility of αD, αF and ASL generates a large opening in PARP2, which could allow exchange of NAD+. NAD+ (yellow) was modelled based on an alignment with PDB 6BHV. d, Regions showing increase in hydrogen–deuterium exchange upon binding of PARP1 to damaged DNA are shown in red. e, Comparison of the activated PARP2–HPF1 (violet and magenta) and open state 1 PARP2–HPF1 (green). Dislocation of PARP2 helices αF, αD and active site loop (ASL) opens the active site for NAD+ binding. PARP2 helices that are not visible in the PARP2–HPF1 structure in open state 1 are indicated in violet on the right. f, As in e but close-up view at NAD+-binding site. PARP2 helices that are not visible in the PARP2–HPF1 structure in open state 1 are shown in violet.
Extended Data Fig. 9 PARP2–HPF1 in open state 2.
a, Model of the disordered H3 N-terminal tail bound to the active site of the PARP2–HPF1–nucleosome complex. The H3 tail can reach the composite active site formed by PARP2 and HPF1 (transparent blue). b, Model of activated PARP2–HPF1 bound to nucleosome (Fig. 1a) was rigid body fitted into the cryo-EM map of PARP2–HPF1 in open state 2 (Extended Data Fig. 3d). PARP2 and HPF1 secondary structure elements are resolved in the cryo-EM map, and the model can be fitted as rigid body. Model is shown in blue and the cryo-EM map in transparent blue. c, Model of PARP2–HPF1 from b is shown fitted into the cryo-EM map. Several PARP2 and HPF1 helices are flexible in this conformation and are not visible in the cryo-EM map. d, NAD+ with the cryo-EM map. In this conformation the NAD+-binding site is closed. NAD+ was modelled based on an alignment with PDB 6BHV. e, Comparison of activated PARP2–HPF1 (violet and magenta) and open state 2 PARP2–HPF1 (blue). Dislocation of PARP2 helices αD and αB and HPF1 helices α7 and α8 opens a potential substrate-release pocket. f, As in e but close-up view at the NAD+-binding site. PARP2 helices that are not visible in the PARP2–HPF1 structure in open state 2 are shown in violet.
Extended Data Fig. 10 Mutations that cause resistance to PARP inhibitors.
a, Linker histone H1 is accessible for PARylation while bound to chromatin. Superposition of cryo-EM models of H1-bound nucleosome (grey; PDB 5NL0) and PARP2–HPF1–nucleosome shows that both complexes can be bound simultaneously to the nucleosome. b, Model of DNA break recognition by PARP enzymes. Environmental sources and errors in DNA processing enzymes can result in DNA breaks. Poly-ADP ribosylation, a post-translation modification deposited by PARP family of enzymes, is the signalling molecule for DNA repair. PARP2 will bind DNA breaks and bridge the two broken ends. This changes the conformation of the autoinhibitory HD subdomain and activates the enzyme to ADP-ribosylate histones. ADP-ribosylation recruits subsequent proteins involved in DNA repair, while PARP–HPF1 remains bound to chromatin. A further increase in PARP automodification releases the complex from chromatin, handing over the repair site to a new set of factors. c, Model of the PARP catalytic cycle. Binding and bridging of a DNA break induces conformational changes that activate PARP proteins, enabling HPF1 binding. In the first step, the NAD+ channel needs to open to bind NAD+. NAD+ binding closes NAD+ channel, and PARPs can add ADP-ribose to the target residue. After catalytic reaction is completed, the product release channel opens, and product can be released and new substrate can bind. d, Point mutations and cancer-associated single-nucleotide polymorphism variants in PARP1 causing PARP inhibitor resistance are shown as blue sticks.
Supplementary Figure 1
This file contains the original gels.
Supplementary Table 1
A list of oligonucleotides used in the study.
| Conformational changes in PARP2 induced by DNA bridging. A video showing conformational changes between the PARP1 bound to one DNA (PDB 4DQY) and the activated PARP2 bridging two nucleosomes (cryo-EM structure).
Rights and permissions
About this article
Cite this article
Bilokapic, S., Suskiewicz, M.J., Ahel, I. et al. Bridging of DNA breaks activates PARP2–HPF1 to modify chromatin. Nature 585, 609–613 (2020). https://doi.org/10.1038/s41586-020-2725-7
This article is cited by
The synthetic lethality of targeting cell cycle checkpoints and PARPs in cancer treatment
Journal of Hematology & Oncology (2022)
Structural dynamics of DNA strand break sensing by PARP-1 at a single-molecule level
Nature Communications (2022)
Nuclear Tkt promotes ischemic heart failure via the cleaved Parp1/Aif axis
Basic Research in Cardiology (2022)
G4-quadruplex-binding proteins: review and insights into selectivity
Biophysical Reviews (2022)
Dual function of HPF1 in the modulation of PARP1 and PARP2 activities
Communications Biology (2021)
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.