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- PDB-3jaa: HUMAN DNA POLYMERASE ETA in COMPLEX WITH NORMAL DNA AND INCO NUCL... -

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Basic information

Entry
Database: PDB / ID: 3jaa
TitleHUMAN DNA POLYMERASE ETA in COMPLEX WITH NORMAL DNA AND INCO NUCLEOTIDE (NRM)
Components
  • DNA (5'-D(*T*CP*AP*TP*TP*AP*TP*GP*AP*CP*GP*CP*T)-3
  • DNA (5'-D(*TP*AP*GP*CP*GP*TP*CP*AP*T)-3')
  • DNA polymerase eta
KeywordsTRANSFERASE/DNA / POL ETA / POLYMERASE / THYMINE DIMER / CPD / XPV / XERODERMA PIGM VARIANT / DNA DAMAGE / TRANSFERASE-DNA COMPLEX
Function / homology
Function and homology information


response to UV-C / error-free translesion synthesis / DNA synthesis involved in DNA repair / cellular response to UV-C / pyrimidine dimer repair / error-prone translesion synthesis / regulation of DNA repair / replication fork / Termination of translesion DNA synthesis / Translesion Synthesis by POLH ...response to UV-C / error-free translesion synthesis / DNA synthesis involved in DNA repair / cellular response to UV-C / pyrimidine dimer repair / error-prone translesion synthesis / regulation of DNA repair / replication fork / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / response to radiation / HDR through Homologous Recombination (HRR) / site of double-strand break / DNA replication / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / nucleoplasm / nucleus / metal ion binding / cytosol
Similarity search - Function
Ubiquitin-Binding Zinc Finger / DNApol eta/Rev1, HhH motif / DNA polymerase eta, ubiquitin-binding zinc finger / : / Zinc finger UBZ3-type profile. / DNA polymerase, Y-family, little finger domain / impB/mucB/samB family C-terminal domain / UmuC domain / DNA polymerase, Y-family, little finger domain superfamily / impB/mucB/samB family ...Ubiquitin-Binding Zinc Finger / DNApol eta/Rev1, HhH motif / DNA polymerase eta, ubiquitin-binding zinc finger / : / Zinc finger UBZ3-type profile. / DNA polymerase, Y-family, little finger domain / impB/mucB/samB family C-terminal domain / UmuC domain / DNA polymerase, Y-family, little finger domain superfamily / impB/mucB/samB family / UmuC domain profile. / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-DZ4 / DNA / DNA (> 10) / DNA polymerase eta
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 22 Å
AuthorsLau, W.C.Y. / Li, Y. / Zhang, Q. / Huen, M.S.Y.
CitationJournal: Sci Rep / Year: 2015
Title: Molecular architecture of the Ub-PCNA/Pol η complex bound to DNA.
Authors: Wilson C Y Lau / Yinyin Li / Qinfen Zhang / Michael S Y Huen /
Abstract: Translesion synthesis (TLS) is the mechanism by which DNA polymerases replicate through unrepaired DNA lesions. TLS is activated by monoubiquitination of the homotrimeric proliferating cell nuclear ...Translesion synthesis (TLS) is the mechanism by which DNA polymerases replicate through unrepaired DNA lesions. TLS is activated by monoubiquitination of the homotrimeric proliferating cell nuclear antigen (PCNA) at lysine-164, followed by the switch from replicative to specialized polymerases at DNA damage sites. Pol η belongs to the Y-Family of specialized polymerases that can efficiently bypass UV-induced lesions. Like other members of the Y-Family polymerases, its recruitment to the damaged sites is mediated by the interaction with monoubiquitinated PCNA (Ub-PCNA) via its ubiquitin-binding domain and non-canonical PCNA-interacting motif in the C-terminal region. The structural determinants underlying the direct recognition of Ub-PCNA by Pol η, or Y-Family polymerases in general, remain largely unknown. Here we report a structure of the Ub-PCNA/Pol η complex bound to DNA determined by single-particle electron microscopy (EM). The overall obtained structure resembles that of the editing PCNA/PolB complex. Analysis of the map revealed the conformation of ubiquitin that binds the C-terminal domain of Pol η. Our present study suggests that the Ub-PCNA/Pol η interaction requires the formation of a structured binding interface, which is dictated by the inherent flexibility of Ub-PCNA.
History
DepositionMay 19, 2015Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Dec 23, 2015Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / Database references / Other
Category: atom_sites / cell ...atom_sites / cell / database_2 / em_image_scans
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[1][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[1][2] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.2Dec 18, 2019Group: Data collection / Database references / Category: em_software / struct_ref_seq_dif
Item: _em_software.fitting_id / _em_software.image_processing_id / _struct_ref_seq_dif.details
Revision 1.3Mar 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Experimental preparation / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_vitrification / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _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_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 / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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Assembly

Deposited unit
A: DNA polymerase eta
T: DNA (5'-D(*T*CP*AP*TP*TP*AP*TP*GP*AP*CP*GP*CP*T)-3
P: DNA (5'-D(*TP*AP*GP*CP*GP*TP*CP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,87213
Polymers55,2903
Non-polymers1,58210
Water7,242402
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 1 molecules A

#1: Protein DNA polymerase eta / RAD30 homolog A / Xeroderma pigmentosum variant type protein


Mass: 48617.707 Da / Num. of mol.: 1 / Fragment: CATALYTIC CORE (RESIDUES 1-432)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLH, RAD30, RAD30A, XPV / Plasmid: PET28 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3)PLYSS / References: UniProt: Q9Y253, DNA-directed DNA polymerase

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DNA chain , 2 types, 2 molecules TP

#2: DNA chain DNA (5'-D(*T*CP*AP*TP*TP*AP*TP*GP*AP*CP*GP*CP*T)-3


Mass: 3941.584 Da / Num. of mol.: 1 / Fragment: DNA TEMPLATE / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*TP*AP*GP*CP*GP*TP*CP*AP*T)-3')


Mass: 2730.810 Da / Num. of mol.: 1 / Fragment: DNA PRIMER / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 412 molecules

#4: Chemical ChemComp-DZ4 / 2'-deoxy-5'-O-[(R)-hydroxy{[(R)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]adenosine


Mass: 490.197 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O11P3
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 402 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY / Number of used crystals: 1
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ub-PCNA/Pol eta/DNA / Type: COMPLEX
Details: Monomeric catalytic core of Pol eta binds to one homotrimeric Ub-PCNA
Molecular weightValue: 0.2 MDa / Experimental value: YES
Buffer solutionpH: 8 / Details: 50 mM Tris-HCl, 5 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
Details: 50 mM Tris-HCl, 5 mM MgCl2 (Stain Details Grids with adsorbed protein floated on two 20 ul drops of 2% w/v uranyl acetate solution for 30 seconds)
EM stainingType: NEGATIVE / Material: Uranyl Acetate
Specimen supportDetails: Continuous carbon coated copper grids (Ted Pella), glow-discharged for 30 seconds

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Electron microscopy imaging

MicroscopyModel: JEOL 2010 / Date: Nov 12, 2014
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 50000 X / Camera length: 0 mm
Specimen holderSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 18 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1UCSF Chimeramodel fitting
2EMAN23D reconstruction
CTF correctionDetails: Particles
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Projection matching / Resolution: 22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 7330 / Details: (Single particle--Applied symmetry: C1) / Num. of class averages: 227 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: REFINEMENT PROTOCOL--RIGID BODY
Atomic model buildingPDB-ID: 3MR2

3mr2


Accession code: 3MR2 / Source name: PDB / Type: experimental model

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