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- PDB-6x0m: Bridging of double-strand DNA break activates PARP2/HPF1 to modif... -

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Basic information

Entry
Database: PDB / ID: 6x0m
TitleBridging of double-strand DNA break activates PARP2/HPF1 to modify chromatin
Components
  • (DNA (167-MER)) x 2
  • Histone PARylation factor 1
  • Poly [ADP-ribose] polymerase 2
KeywordsGENE REGULATION / DNA repair / PARP1 / PARP2 / HPF1 / ADP-ribosylation / chromatin / histone modifications
Function / homology
Function and homology information


protein ADP-ribosyltransferase-substrate adaptor activity / regulation of protein ADP-ribosylation / response to oxygen-glucose deprivation / hippocampal neuron apoptotic process / poly-ADP-D-ribose binding / positive regulation of cell growth involved in cardiac muscle cell development / NAD+-protein-serine ADP-ribosyltransferase activity / NAD+-histone H2BS6 serine ADP-ribosyltransferase activity / NAD+-histone H3S10 serine ADP-ribosyltransferase activity / NAD DNA ADP-ribosyltransferase activity ...protein ADP-ribosyltransferase-substrate adaptor activity / regulation of protein ADP-ribosylation / response to oxygen-glucose deprivation / hippocampal neuron apoptotic process / poly-ADP-D-ribose binding / positive regulation of cell growth involved in cardiac muscle cell development / NAD+-protein-serine ADP-ribosyltransferase activity / NAD+-histone H2BS6 serine ADP-ribosyltransferase activity / NAD+-histone H3S10 serine ADP-ribosyltransferase activity / NAD DNA ADP-ribosyltransferase activity / NAD+-protein-glutamate ADP-ribosyltransferase activity / DNA ADP-ribosylation / poly-ADP-D-ribose modification-dependent protein binding / NAD+-protein-aspartate ADP-ribosyltransferase activity / HDR through MMEJ (alt-NHEJ) / NAD+-histone H2BE35 glutamate ADP-ribosyltransferase activity / NAD+-histone H2BE2 glutamate ADP-ribosyltransferase activity / NAD+-histone H2BE18 glutamate ADP-ribosyltransferase activity / NAD+ ADP-ribosyltransferase / protein auto-ADP-ribosylation / protein poly-ADP-ribosylation / DNA repair-dependent chromatin remodeling / NAD+-protein mono-ADP-ribosyltransferase activity / site of DNA damage / NAD+ poly-ADP-ribosyltransferase activity / decidualization / Transferases; Glycosyltransferases; Pentosyltransferases / POLB-Dependent Long Patch Base Excision Repair / nucleosome binding / extrinsic apoptotic signaling pathway / nucleotidyltransferase activity / DNA Damage Recognition in GG-NER / base-excision repair / Dual Incision in GG-NER / Formation of Incision Complex in GG-NER / double-strand break repair / histone binding / damaged DNA binding / DNA repair / DNA damage response / chromatin binding / chromatin / nucleolus / nucleoplasm / nucleus
Similarity search - Function
Histone PARylation factor 1 / Histone PARylation factor 1 / : / Poly(ADP-ribose) polymerase, regulatory domain / WGR domain / WGR domain superfamily / WGR domain / WGR domain profile. / Proposed nucleic acid binding domain / Poly(ADP-ribose) polymerase, regulatory domain superfamily ...Histone PARylation factor 1 / Histone PARylation factor 1 / : / Poly(ADP-ribose) polymerase, regulatory domain / WGR domain / WGR domain superfamily / WGR domain / WGR domain profile. / Proposed nucleic acid binding domain / Poly(ADP-ribose) polymerase, regulatory domain superfamily / Poly(ADP-ribose) polymerase, regulatory domain / PARP alpha-helical domain profile. / Poly(ADP-ribose) polymerase catalytic domain / Poly(ADP-ribose) polymerase, catalytic domain / PARP catalytic domain profile.
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone PARylation factor 1 / Poly [ADP-ribose] polymerase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsHalic, M. / Bilokapic, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM135599-01 United States
CitationJournal: Nature / Year: 2020
Title: Bridging of DNA breaks activates PARP2-HPF1 to modify chromatin.
Authors: Silvija Bilokapic / Marcin J Suskiewicz / Ivan Ahel / Mario Halic /
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). In the DNA damage responses, ...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). In the DNA damage responses, this post-translational modification occurs predominantly on serine residues and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine. 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. 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.
History
DepositionMay 16, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 30, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 7, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
O: Histone PARylation factor 1
P: Poly [ADP-ribose] polymerase 2
o: Histone PARylation factor 1
p: Poly [ADP-ribose] polymerase 2
I: DNA (167-MER)
J: DNA (167-MER)
i: DNA (167-MER)
j: DNA (167-MER)


Theoretical massNumber of molelcules
Total (without water)421,5558
Polymers421,5558
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10160 Å2
ΔGint-38 kcal/mol
Surface area73420 Å2

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Components

#1: Protein Histone PARylation factor 1


Mass: 40598.293 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HPF1, C4orf27 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9NWY4
#2: Protein Poly [ADP-ribose] polymerase 2 / hPARP-2 / ADP-ribosyltransferase diphtheria toxin-like 2 / ARTD2 / DNA ADP-ribosyltransferase PARP2 ...hPARP-2 / ADP-ribosyltransferase diphtheria toxin-like 2 / ARTD2 / DNA ADP-ribosyltransferase PARP2 / NAD(+) ADP-ribosyltransferase 2 / ADPRT-2 / Poly[ADP-ribose] synthase 2 / pADPRT-2 / Protein poly-ADP-ribosyltransferase PARP2


Mass: 67072.500 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PARP2, ADPRT2, ADPRTL2 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9UGN5, NAD+ ADP-ribosyltransferase, Transferases; Glycosyltransferases; Pentosyltransferases
#3: DNA chain DNA (167-MER)


Mass: 51776.004 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: DNA chain DNA (167-MER)


Mass: 51330.684 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA ComplexCOMPLEX2xPARP2/HPF1 from PARP2/HPF1_Nucleosome complexall0RECOMBINANT
2Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2COMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Escherichia coli (E. coli)562
12Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector model
1180COUNTINGGATAN K3 BIOQUANTUM (6k x 4k)
2180GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 934000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00712781
ELECTRON MICROSCOPYf_angle_d1.22817438
ELECTRON MICROSCOPYf_dihedral_angle_d22.1684874
ELECTRON MICROSCOPYf_chiral_restr0.0661883
ELECTRON MICROSCOPYf_plane_restr0.0072097

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