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- EMDB-21979: Bridging of double-strand DNA break activates PARP2/HPF1 to modif... -

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Basic information

Entry
Database: EMDB / ID: EMD-21979
TitleBridging of double-strand DNA break activates PARP2/HPF1 to modify chromatin
Map data2xPARP2/HPF1 from PARP2/HPF1_Nucleosome complex
Sample
  • Complex: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
    • Complex: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2
      • Protein or peptide: Histone PARylation factor 1
      • Protein or peptide: Poly [ADP-ribose] polymerase 2
    • Complex: DNA
      • DNA: DNA (167-MER)
      • DNA: DNA (167-MER)
KeywordsDNA repair / PARP1 / PARP2 / HPF1 / ADP-ribosylation / chromatin / histone modifications / GENE REGULATION
Function / homology
Function and homology information


protein ADP-ribosyltransferase-substrate adaptor activity / hippocampal neuron apoptotic process / response to oxygen-glucose deprivation / regulation of protein ADP-ribosylation / poly-ADP-D-ribose binding / positive regulation of cell growth involved in cardiac muscle cell development / NAD+-protein-serine ADP-ribosyltransferase activity / NAD DNA ADP-ribosyltransferase activity / NAD+- protein-aspartate ADP-ribosyltransferase activity / NAD+-protein-glutamate ADP-ribosyltransferase activity ...protein ADP-ribosyltransferase-substrate adaptor activity / hippocampal neuron apoptotic process / response to oxygen-glucose deprivation / regulation of protein ADP-ribosylation / poly-ADP-D-ribose binding / positive regulation of cell growth involved in cardiac muscle cell development / NAD+-protein-serine ADP-ribosyltransferase activity / NAD DNA ADP-ribosyltransferase activity / NAD+- protein-aspartate ADP-ribosyltransferase activity / NAD+-protein-glutamate ADP-ribosyltransferase activity / DNA ADP-ribosylation / HDR through MMEJ (alt-NHEJ) / poly-ADP-D-ribose modification-dependent protein binding / DNA repair-dependent chromatin remodeling / NAD+ ADP-ribosyltransferase / protein auto-ADP-ribosylation / protein poly-ADP-ribosylation / site of DNA damage / NAD+-protein ADP-ribosyltransferase activity / decidualization / NAD+ ADP-ribosyltransferase activity / Transferases; Glycosyltransferases; Pentosyltransferases / nucleosome binding / POLB-Dependent Long Patch Base Excision Repair / extrinsic apoptotic signaling pathway / nucleotidyltransferase activity / DNA Damage Recognition in GG-NER / Dual Incision in GG-NER / base-excision repair / 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 / WGR domain profile. / Poly(ADP-ribose) polymerase, regulatory domain / WGR domain / WGR domain superfamily / WGR domain / Proposed nucleic acid binding domain / Poly(ADP-ribose) polymerase, regulatory domain superfamily / Poly(ADP-ribose) polymerase, regulatory domain ...Histone PARylation factor 1 / Histone PARylation factor 1 / WGR domain profile. / Poly(ADP-ribose) polymerase, regulatory domain / WGR domain / WGR domain superfamily / WGR domain / 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
Histone PARylation factor 1 / Poly [ADP-ribose] polymerase 2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsHalic M / Bilokapic S
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseSep 16, 2020-
Map releaseSep 16, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6x0m
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21979.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2xPARP2/HPF1 from PARP2/HPF1_Nucleosome complex
Voxel sizeX=Y=Z: 2.12 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.07
Minimum - Maximum-0.10209225 - 0.29856485
Average (Standard dev.)0.0001796896 (±0.010857495)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 508.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z508.800508.800508.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1020.2990.000

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Supplemental data

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Sample components

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Entire : Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA C...

EntireName: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
Components
  • Complex: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
    • Complex: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2
      • Protein or peptide: Histone PARylation factor 1
      • Protein or peptide: Poly [ADP-ribose] polymerase 2
    • Complex: DNA
      • DNA: DNA (167-MER)
      • DNA: DNA (167-MER)

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Supramolecule #1: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA C...

SupramoleculeName: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: 2xPARP2/HPF1 from PARP2/HPF1_Nucleosome complex
Molecular weightTheoretical: 100 KDa

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Supramolecule #2: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2

SupramoleculeName: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Histone PARylation factor 1

MacromoleculeName: Histone PARylation factor 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.598293 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGG MVGGGGKRRP GGEGPQCEKT TDVKKSKFCE ADVSSDLRKE VENHYKLSLP EDFYHFWKFC EELDPEKPSD SLSASLGLQ LVGPYDILAG KHKTKKKSTG LNFNLHWRFY YDPPEFQTII IGDNKTQYHM GYFRDSPDEF PVYVGINEAK K NCIIVPNG ...String:
MGHHHHHHGG MVGGGGKRRP GGEGPQCEKT TDVKKSKFCE ADVSSDLRKE VENHYKLSLP EDFYHFWKFC EELDPEKPSD SLSASLGLQ LVGPYDILAG KHKTKKKSTG LNFNLHWRFY YDPPEFQTII IGDNKTQYHM GYFRDSPDEF PVYVGINEAK K NCIIVPNG DNVFAAVKLF LTKKLKEITD KKKINLLKNI DEKLTEAARE LGYSLEQRTV KMKQRDKKVV TKTFHGAGLV VP VDKNDVG YRELPETDAD LKRICKTIVE AASDEERLKA FAPIQEMMTF VQFANDECDY GMGLELGMDL FCYGSHYFHK VAG QLLPLA YNLLKRNLFA EIIEEHLANR SQENIDQLAA

UniProtKB: Histone PARylation factor 1

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Macromolecule #2: Poly [ADP-ribose] polymerase 2

MacromoleculeName: Poly [ADP-ribose] polymerase 2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: NAD+ ADP-ribosyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.0725 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MAARRRRSTG GGRARALNES KRVNNGNTAP EDSSPAKKTR RCQRQESKKM PVAGGKANKD RTEDKQDES VKALLLKGKA PVDPECTAKV GKAHVYCEGN DVYDVMLNQT NLQFNNNKYY LIQLLEDDAQ RNFSVWMRWG R VGKMGQHS ...String:
MGSSHHHHHH SSGLVPRGSH MAARRRRSTG GGRARALNES KRVNNGNTAP EDSSPAKKTR RCQRQESKKM PVAGGKANKD RTEDKQDES VKALLLKGKA PVDPECTAKV GKAHVYCEGN DVYDVMLNQT NLQFNNNKYY LIQLLEDDAQ RNFSVWMRWG R VGKMGQHS LVACSGNLNK AKEIFQKKFL DKTKNNWEDR EKFEKVPGKY DMLQMDYATN TQDEEETKKE ESLKSPLKPE SQ LDLRVQE LIKLICNVQA MEEMMMEMKY NTKKAPLGKL TVAQIKAGYQ SLKKIEDCIR AGQHGRALME ACNEFYTRIP HDF GLRTPP LIRTQKELSE KIQLLEALGD IEIAIKLVKT ELQSPEHPLD QHYRNLHCAL RPLDHESYEF KVISQYLQST HAPT HSDYT MTLLDLFEVE KDGEKEAFRE DLHNRMLLWH GSRMSNWVGI LSHGLRIAPP EAPITGYMFG KGIYFADMSS KSANY CFAS RLKNTGLLLL SEVALGQCNE LLEANPKAEG LLQGKHSTKG LGKMAPSSAH FVTLNGSTVP LGPASDTGIL NPDGYT LNY NEYIVYNPNQ VRMRYLLKVQ FNFLQLW

UniProtKB: Poly [ADP-ribose] polymerase 2

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Macromolecule #3: DNA (167-MER)

MacromoleculeName: DNA (167-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 51.776004 KDa
SequenceString: (DC)(DA)(DA)(DT)(DA)(DC)(DA)(DT)(DG)(DC) (DA)(DC)(DA)(DG)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA) (DC)(DA)(DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG) (DG) (DA)(DG)(DA)(DC)(DT)(DA) ...String:
(DC)(DA)(DA)(DT)(DA)(DC)(DA)(DT)(DG)(DC) (DA)(DC)(DA)(DG)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA) (DC)(DA)(DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG) (DG) (DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG) (DG)(DA)(DG)(DT)(DA)(DA)(DT)(DC)(DC)(DC) (DC)(DT) (DT)(DG)(DG)(DC)(DG)(DG)(DT) (DT)(DA)(DA)(DA)(DA)(DC)(DG)(DC)(DG)(DG) (DG)(DG)(DG) (DA)(DC)(DA)(DG)(DC)(DG) (DC)(DG)(DT)(DA)(DC)(DG)(DT)(DG)(DC)(DG) (DT)(DT)(DT)(DA) (DA)(DG)(DC)(DG)(DG) (DT)(DG)(DC)(DT)(DA)(DG)(DA)(DG)(DC)(DT) (DG)(DT)(DC)(DT)(DA) (DC)(DG)(DA)(DC) (DC)(DA)(DA)(DT)(DT)(DG)(DA)(DG)(DC)(DG) (DG)(DC)(DC)(DT)(DC)(DG) (DG)(DC)(DA) (DC)(DC)(DG)(DG)(DG)(DA)(DT)(DT)(DC)(DT) (DC)(DC)(DA)(DG)(DG)(DG)(DC) (DA)(DT) (DC)(DA)(DT)(DA)(DG)

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Macromolecule #4: DNA (167-MER)

MacromoleculeName: DNA (167-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 51.330684 KDa
SequenceString: (DC)(DT)(DA)(DT)(DG)(DA)(DT)(DG)(DC)(DC) (DC)(DT)(DG)(DG)(DA)(DG)(DA)(DA)(DT)(DC) (DC)(DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG) (DA)(DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA) (DA) (DT)(DT)(DG)(DG)(DT)(DC) ...String:
(DC)(DT)(DA)(DT)(DG)(DA)(DT)(DG)(DC)(DC) (DC)(DT)(DG)(DG)(DA)(DG)(DA)(DA)(DT)(DC) (DC)(DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG) (DA)(DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA) (DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT) (DA)(DG)(DA)(DC)(DA)(DG)(DC)(DT)(DC)(DT) (DA)(DG) (DC)(DA)(DC)(DC)(DG)(DC)(DT) (DT)(DA)(DA)(DA)(DC)(DG)(DC)(DA)(DC)(DG) (DT)(DA)(DC) (DG)(DC)(DG)(DC)(DT)(DG) (DT)(DC)(DC)(DC)(DC)(DC)(DG)(DC)(DG)(DT) (DT)(DT)(DT)(DA) (DA)(DC)(DC)(DG)(DC) (DC)(DA)(DA)(DG)(DG)(DG)(DG)(DA)(DT)(DT) (DA)(DC)(DT)(DC)(DC) (DC)(DT)(DA)(DG) (DT)(DC)(DT)(DC)(DC)(DA)(DG)(DG)(DC)(DA) (DC)(DG)(DT)(DG)(DT)(DC) (DA)(DG)(DA) (DT)(DA)(DT)(DA)(DT)(DA)(DC)(DA)(DT)(DC) (DC)(DT)(DG)(DT)(DG)(DC)(DA) (DT)(DG) (DT)(DA)(DT)(DT)(DG)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Detector mode: COUNTING / #0 - Average electron dose: 80.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Average electron dose: 80.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 934000
Startup modelType of model: EMDB MAP
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 16000
Image recording ID1

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