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- PDB-6p1h: Cryo-EM Structure of DNA Polymerase Delta Holoenzyme -

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Basic information

Entry
Database: PDB / ID: 6p1h
TitleCryo-EM Structure of DNA Polymerase Delta Holoenzyme
Components
  • (DNA polymerase delta ...) x 3
  • DNA (30-MER)
Keywordsdna binding protein/dna / DNA binding / enzyme / catalysis / regulation / DNA BINDING PROTEIN / dna binding protein-dna complex
Function / homology
Function and homology information


delta DNA polymerase complex / DNA-templated DNA replication maintenance of fidelity / DNA amplification / RNA-templated DNA biosynthetic process / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA replication, removal of RNA primer / lagging strand elongation / double-strand break repair via break-induced replication / postreplication repair ...delta DNA polymerase complex / DNA-templated DNA replication maintenance of fidelity / DNA amplification / RNA-templated DNA biosynthetic process / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA replication, removal of RNA primer / lagging strand elongation / double-strand break repair via break-induced replication / postreplication repair / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / 3'-5'-DNA exonuclease activity / DNA metabolic process / DNA strand elongation involved in DNA replication / leading strand elongation / mismatch repair / base-excision repair, gap-filling / replication fork / nucleotide-excision repair / base-excision repair / DNA-templated DNA replication / 4 iron, 4 sulfur cluster binding / DNA replication / molecular adaptor activity / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / nucleus / metal ion binding / cytosol
Similarity search - Function
DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B signature. ...DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA polymerase delta catalytic subunit / DNA polymerase delta small subunit / DNA polymerase delta subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJain, R. / Rice, W. / Aggarwal, A.K.
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Cryo-EM structure and dynamics of eukaryotic DNA polymerase δ holoenzyme.
Authors: Rinku Jain / William J Rice / Radhika Malik / Robert E Johnson / Louise Prakash / Satya Prakash / Iban Ubarretxena-Belandia / Aneel K Aggarwal /
Abstract: DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. ...DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe-4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.
History
DepositionMay 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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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-20235
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA polymerase delta catalytic subunit
B: DNA polymerase delta small subunit
C: DNA polymerase delta subunit 3
P: DNA (30-MER)
T: DNA (30-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)243,31812
Polymers242,2995
Non-polymers1,0197
Water79344
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16330 Å2
ΔGint-147 kcal/mol
Surface area66540 Å2
MethodPISA

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Components

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DNA polymerase delta ... , 3 types, 3 molecules ABC

#1: Protein DNA polymerase delta catalytic subunit / DNA polymerase III


Mass: 127438.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: POL3, CDC2, TEX1, YDL102W, D2366 / Variant: ATCC 204508 / S288c / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P15436, DNA-directed DNA polymerase
#2: Protein DNA polymerase delta small subunit / Hydroxyurea-sensitive protein 2


Mass: 55987.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: POL31, HUS2, HYS2, SDP5, YJR006W, J1427, YJR83.7 / Variant: ATCC 204508 / S288c / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P46957, DNA-directed DNA polymerase
#3: Protein DNA polymerase delta subunit 3


Mass: 40377.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: POL32, YJR043C, J1626 / Variant: ATCC 204508 / S288c / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P47110

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DNA chain , 1 types, 2 molecules PT

#4: DNA chain DNA (30-MER)


Mass: 9247.966 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 51 molecules

#5: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#6: Chemical ChemComp-DCP / 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE


Mass: 467.157 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C9H16N3O13P3
#7: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 44 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: enzymatic complex with ligands / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.220 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Buffer solutionpH: 6.8
SpecimenConc.: 1.2 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 recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
7PHENIXmodel fitting
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166444 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00913288
ELECTRON MICROSCOPYf_angle_d0.76118123
ELECTRON MICROSCOPYf_dihedral_angle_d10.6297931
ELECTRON MICROSCOPYf_chiral_restr0.052029
ELECTRON MICROSCOPYf_plane_restr0.0052223

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