Bridging of DNA breaks activates PARP2-HPF1 to modify chromatin

Abstract

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)^1-5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues^6-8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine^9,10. 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 breaks^2,11. 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 treatments^12-16.

Extended Data Fig. 1.. Assembly and cryo-EM of PARP2/HPF1 bound to mono-nucleosomes.

a) SDS-PAGE showing the PARP2/HPF1_Nucleosome complex assembly for cryo-EM. b) Native gel showing the PARP2/HPF1_Nucleosome complex assembly for cryo-EM. Note the shift in the PARP2_Nucleosome complex migration upon binding of HPF1. c) SDS-PAGE/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 nuclesomes 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 dataset comprising 934 000 particles. The dataset was further extensively classified. The regions used for focused classifications and refinements in b-g are color coded and labeled. 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 colored 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 shown 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 colored 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 shown on the right. f) Focused classification and refinements with focus on connection of the PARP2_WGR domains with the 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 shown 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 shown on the right. g) Focused classification and refinements with focus on connection of the PARP2_WGR domains with the Nucleosome 1 (see e). Cryo-EM map of this region was refined to 5.7 Å (left). FSC curve showing the resolution of the map is shown 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 shown on the right.

Extended Data Fig. 3.. Focused classification, refinement and model building: focus on the PARP2/HPF1 complex.

a) The overall dataset comprising 934 000 particles was extensively classified with focus on PARP2/HPF1 complex found in between 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 shown below. c) Cryo-EM map of PARP2/HPF1 bound to the nucleosome in the open state 1. Cryo-EM map of this conformation was refined to 6.7 Å. FSC curve showing the resolution of the map is shown below. The map is colored by local resolution. d) Cryo-EM map of PARP2/HPF1 bound to the nucleosome in the 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 colored 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 colored by local resolution. f) Angular distribution for PARP2/HPF1. g) Directional FSC plot showing reasonably uniform resolution in all directions. h) The model of the PARP2_WGR domain (PDB:6F5B), PARP2 catalytic domain (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. Final map containing two PARP2/HPF1 (middle) was refined to 6.3 Å. FSC curve is shown on the right. The map is colored 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 mono-nucleosomes. c) Schematic representation of PARP2 and HPF1 organization is shown on the left. On the right PARP2/HPF1 model is colored by domains. d) Map quality and fit of the model is shown for the region shown in Fig. 1d. e) Alignment of PARP2/HPF1 bridging two nucleosomes with 5’-phosphate 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’-phosphate (yellow, PDB:6F5B). f) Alignment of PARP2/HPF1 bridging two nucleosomes with 5’-phosphate DNA with the X-ray structure of isolated PARP2 WGR domains bound to double-strand DNA with 5’-phosphate. The model is colored by RMSD. g) Map quality and fit of the model are shown for the regions shown in Fig. 1e.

Extended Data Fig. 5.. HPF1 interaction 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 labeled with Alexa 488. PARP2/HPF1 binds nucleosomes with higher affinity than free DNA. b) PARP2_Nucleosome and PARP2/HPF1_Nucleosome complex assembly analyzed by EMSA. 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) PARP2/HPF1_Nucleosome complex assembly with wild type and mutant HPF1 analyzed by EMSA. Mutations in loops that interact with nucleosomal DNA destabilize the complex. Native gel is stained with SYBR Gold. e) One of 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 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 (gray, PDB:6TX3) and the PARP2/HPF1_Nucleosome cryo-EM model (violet and magenta). HPF1 slightly rearranges in the cryo-EM structure when compared to the X-ray structure. g) Superposition of the PARP2 catalytic domains from the PARP2/HPF1 crystal structure (gray, PDB:6TX3) and the PARP2/HPF1_Nucleosome cryo-EM model. The model is colored by RMSD. 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 two nucleosomes is required for PARP2 activation.

a) PARP2 can bind to double-stranded DNA with 5’-biotin on both ends but can not bridge that DNA. The complex formation was followed on the native gel by staining with SYBR Gold and anti-PARP2 western blot. 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 with *. c) Lanes with equal amounts of PARP2 bridging two DNA from b) (marked with *) were incubated with NAD⁺ for 7 min to perform in-gel PARylation assay. 2.5x 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) marked with +. 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 mutant PARP2 proteins. f) SDS-PAGE showing PARP2 auto-PARylation activity. Wild type and mutant PARP2 were incubated with NAD⁺ and with NAD⁺ and DNA. Note increased DNA-independent activity of PARP2 mutants, and reduction in DNA-dependent activity. g) Native gel and anti-H3 Western showing the PARP2_Nucleosome and PARP2/HPF1_Nucleosome complex assembly with wild type and mutant PARP2. Mutations in PARP2 R140 that bridges two nucleosomes abolishes complex formation. Mutation in 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 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 (gray, PDB: 4DQY). Note conformational changes in the CAT_HD domain, especially in helices αA, αB, αF and αG. b) Alignment of WGR domains of PARP2 cryo-EM model (violet) and PARP1 DNA bound X-ray structure (gray, PDB: 4DQY). The model is colored by RMSD. 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 (gray, PDB: 4TVJ). Note conformational changes in αB, and the hydrophobic and HD loops. d) Close-up view of the PARP2 WGR signaling 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 labeled. f) Point mutations in PARP1 and PARP2 showing increase in DNA-independent activity are labeled as red sticks. g) The map and the fit of the model are shown for the NAD⁺ binding site.

Extended Data Fig. 8.. Model for the PARP2/HPF1 in the open state 1.

a) Model of activated PARP2/HPF1 bound to nucleosome (Fig. 1a) was rigid body fitted into the cryo-EM map of 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 NAD⁺ binding site. Flexibility of αD, αF and ASL generates large opening in PARP2, which could allow exchange of NAD⁺. NAD⁺ (yellow) was modeled based on an alignment with PDB:6BHV. d) Regions showing increase in hydrogen-deuterium exchange upon PARP1 binding to damaged DNA are shown in red. e) Comparison of the activated PARP2/HPF1 (violet/magenta) and the 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 the open state 1 are shown 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 the open state 1 are shown in violet.

Extended Data Fig. 9.. PARP2/HPF1 in the 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 the activated PARP2/HPF1 bound to nucleosome (Fig. 1a) was rigid body fitted into the cryo-EM map of PARP2/HPF1 in the 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⁺ is shown with the cryo-EM map. In this conformation NAD⁺ binding site is closed. NAD⁺ was modeled based on an alignment with PDB:6BHV. e) Comparison of the activated PARP2/HPF1 (violet/magenta) and the open state 2 PARP2/HPF1 (blue). Dislocation of PARP2 helices αD and αB, and HPF1 helices α7 and α8, opens potential substrate release pocket. 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 2 are shown in violet.

Extended Data Fig. 10.. Mutation 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 (gray, PDB:5NL0) and PARP2/HPF1_Nucleosome shows that both complexes can be simultaneously bound 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, post-translation modification deposited by PARP family of enzymes, is the signaling molecule for DNA repair. PARP2 will bind DNA breaks and bridge 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. Further increase in PARP automodification releases the complex from chromatin, handing over the repair site to new set of factors. c) Model of PARP catalytic cycle. Binding and bridging of DNA break induces conformational changes that activate PARP, enabling HPF1 binding. In the first step, the NAD⁺ channel needs to open to bind NAD⁺. NAD⁺ binding closes NAD⁺ channel and PARP can add ADP-ribose to target residue. After catalytic reaction is completed, product release channel opens, and product can be released and new substrate can bind. d) Point mutations and cancer associated SNP variants in PARP1 causing PARP inhibitor resistance are shown as blue sticks.

Fig. 1.. PARP2/HPF1 bridges two mono-nucleosomes.

a) Composite cryo-EM map of PARP2/HPF1 bound to 5’-phosphorylated nucleosome at 2.1–3.9 Å resolution. b) Model for the cryo-EM structure of PARP2/HPF1 bound to bridged mono-nucleosomes. c) Model for the cryo-EM map with two PARP2/HPF1 bound to bridged mono-nucleosomes. Two PARP2/HPF1 are positioned to modify H3 on opposite sides of nucleosomes. d) View at the bridge with two PARP2 WGR domains connecting two nucleosomes containing a double-strand DNA break. Two DNAs are positioned in a way that the 5’P of the Nucleosome 1 DNA is aligned with the 3’OH group of the Nucleosome 2 DNA, and vice versa. PARP2 catalytic domain binds the linker DNA with the loop in the HD subdomain (CAT_HD). HPF1 binds the linker DNA through the helix α8. e) HPF1 binds nucleosomal DNA near the dyad with several positively charged residues in the N-terminal domain.

Fig. 2.. Bridging of DNA break activates PARP2.

a) In-gel PARylation assay. PARP2 was bound to DNA and distinct complexes were separated: PARP2 bound to one DNA, one PARP2 bridging two DNAs and two PARP2 bridging two DNAs (note stronger PARP2 signal in the upper band). Gel was incubated with NAD⁺ and ADP-ribosylation was detected. b) Activity of wild type and mutant PARP2 on the nucleosome detected by SDS-PAGE/immunoblotting. c) Structural alignment of WGR domains of the nucleosome bound PARP2 (violet) and the DNA bound PARP1 (gray, PDB: 4DQY). The signaling loop, as in PARP1_WGR, would clash with the second DNA (red mark). d) Close-up view of the signaling loop (aa:139–145) interactions with DNA, the hydrophobic loop (aa:248–258) and the HD loop (aa:297–310). e) Close-up view of the PARP2 hydrophobic pocket formed by the hydrophobic loop, the signaling loop and the HD loop. V141 in the signaling loop interacts with L254 and P253 of the hydrophobic loop. f) Close-up view of the nucleosome bound PARP2 (violet) superimposed to the PARP2 crystal structure (gray, PDB:4TVJ). Rearrangement of the HD subdomain alleviates the steric clash with NAD⁺. g) X-ray structure of the PARP2 catalytic domain (gray, PDB:4TVJ) was superimposed on the cryo-EM structure of PARP2/HPF1. HPF1 clashes with the PARP2 E244 in its inactive conformation (gray, PDB:4TVJ). Rearrangement of PARP2 αB enables HPF1 binding. h) In-gel PARylation assay as in a): HPF1 binds only PARP2 that bridged two DNAs. One representative of at least 3 independent biochemical experiments is shown. For gel source data, see Supplementary Fig. 1.

Fig. 3.. PARP2 catalytic domain rearranges to open NAD⁺ and substrate binding sites.

a) View at the active site formed by PARP2 and HPF1 in the nucleosome bound activated conformation. b) View at the active site formed by PARP2 and HPF1 in the nucleosome bound open state 1. PARP2 helices αF, αD and active site loop (ASL) are flexible, which might open active site for NAD⁺ binding. c) View at the active site formed by PARP2 and HPF1 in the nucleosome bound open state 2. Dislocation of PARP2 helices αD and αB, and HPF1 helices α7 and α8, might open substrate release pocket. NAD⁺ (yellow) was modeled based on an alignment with PDB:6BHV.

Fig. 4.. PARylated PARP2/HPF1 dissociate from the chromatin.

a) Assembled PARP2/HPF1_Nucleosome complex was incubated with and without NAD⁺ and analyzed by EMSA. Note the shift in the position of PARylated nucleosomes in respect to the unmodified nucleosomes. PARylated PARP2 does not co-migrate with PARylated nucleosomes, indicating dissociation. b) SDS-PAGE/immunoblotting of the reaction in a). The complex activation leads to ADP-ribosylation of H3. c) The changes in the PARP2/HPF1_Nucleosome complex during PARylation reaction were followed by EMSA. Initial PARylation does not lead to the complex dissociation; however, longer PARylation dissociates the complex and generates PARylated nucleosomes. d) The extent of the PARylation reaction was followed by PAGE/immunoblotting. In the first steps, H3, HPF1 and PARP2 are PARylated to similar extent. Extended reaction (60 min) leads to increase in PARP2 auto-PARylation. One representative of at least 3 independent biochemical experiments is shown. For gel source data, see Supplementary Fig. 1.