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- PDB-6wz9: Bridging of double-strand DNA break activates PARP2/HPF1 to modif... -

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Basic information

Entry
Database: PDB / ID: 6wz9
TitleBridging of double-strand DNA break activates PARP2/HPF1 to modify chromatin
Components
  • (DNA) x 2
  • Histone H2A
  • Histone H2B 1.1
  • Histone H3.2
  • Histone H4
KeywordsGENE REGULATION / DNA repair / PARP1 / PARP2 / HPF1 / ADP-ribosylation / chromatin / histone modifications
Function / homology
Function and homology information


DNA-templated transcription, initiation / nucleosome / protein heterodimerization activity / DNA binding / nucleoplasm / nucleus
Similarity search - Function
Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A signature. / Histone H2A conserved site / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / Histone H4 signature. ...Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A signature. / Histone H2A conserved site / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / Histone H4 signature. / Histone H4, conserved site / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF) / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Core histone H2A/H2B/H3/H4 / Histone H2A/H2B/H3 / Histone-fold
Similarity search - Domain/homology
DNA (> 100) / DNA (> 10) / DNA / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Histone H2A
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 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 13, 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

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-21971
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A
D: Histone H2B 1.1
E: Histone H3.2
F: Histone H4
G: Histone H2A
H: Histone H2B 1.1
I: DNA
J: DNA


Theoretical massNumber of molelcules
Total (without water)211,24710
Polymers211,24710
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area55980 Å2
ΔGint-384 kcal/mol
Surface area70930 Å2

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3.2


Mass: 15303.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P84233
#2: Protein Histone H4 /


Mass: 11263.231 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P62799
#3: Protein Histone H2A /


Mass: 13978.241 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: hist1h2aj, h2ac14, LOC494591, XELAEV_18003602mg / Production host: Escherichia coli (E. coli) / References: UniProt: Q6AZJ8, UniProt: P06897*PLUS
#4: Protein Histone H2B 1.1 / H2B1.1


Mass: 13524.752 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P02281

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA /


Mass: 51776.004 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#6: DNA chain DNA /


Mass: 51330.684 Da / Num. of mol.: 1
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 H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1/DNA ComplexCOMPLEXNucleosome 1 from PARP2/HPF1_Nucleosome complex#1-#60RECOMBINANT
2Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1 2COMPLEX#1-#41RECOMBINANT
3DNACOMPLEX#5-#61RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Xenopus laevis (African clawed frog)8355
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
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 FIELDBright-field microscopy
Image recordingElectron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN 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
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 934000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00612841
ELECTRON MICROSCOPYf_angle_d0.72318637
ELECTRON MICROSCOPYf_dihedral_angle_d29.7415288
ELECTRON MICROSCOPYf_chiral_restr0.0432127
ELECTRON MICROSCOPYf_plane_restr0.0051317

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