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- PDB-8ef9: Structure of Lates calcarifer DNA polymerase theta polymerase dom... -

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Basic information

Entry
Database: PDB / ID: 8ef9
TitleStructure of Lates calcarifer DNA polymerase theta polymerase domain with long duplex DNA, complex Ia
Components
  • DNA (5'-D(*AP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(2DA))-3')
  • DNA (5'-D(*AP*GP*CP*TP*CP*TP*AP*CP*GP*GP*AP*TP*GP*C)-3')
  • DNA polymerase theta
KeywordsDNA BINDING PROTEIN/DNA / DNA double-strand break repair / Microhomology-mediated end joining / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10)
Function and homology information
Biological speciesLates calcarifer (barramundi perch)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsLi, C. / Zhu, H. / Sun, J. / Gao, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
American Cancer SocietyRSG-22-082-01-DMC United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR170062 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural basis of DNA polymerase θ mediated DNA end joining.
Authors: Chuxuan Li / Hanwen Zhu / Shikai Jin / Leora M Maksoud / Nikhil Jain / Ji Sun / Yang Gao /
Abstract: DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes ...DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.
History
DepositionSep 8, 2022Deposition site: RCSB / Processing site: RCSB
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Revision 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase theta
E: DNA (5'-D(*AP*GP*CP*TP*CP*TP*AP*CP*GP*GP*AP*TP*GP*C)-3')
F: DNA (5'-D(*AP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(2DA))-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,9155
Polymers110,3843
Non-polymers5312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DNA polymerase theta


Mass: 95674.117 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lates calcarifer (barramundi perch)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: DNA chain DNA (5'-D(*AP*GP*CP*TP*CP*TP*AP*CP*GP*GP*AP*TP*GP*C)-3')


Mass: 8864.712 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Lates calcarifer (barramundi perch)
#3: DNA chain DNA (5'-D(*AP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(2DA))-3')


Mass: 5844.796 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Lates calcarifer (barramundi perch)
#4: Chemical ChemComp-DGT / 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Ternary complex of Lates calcarifer DNA polymerase Theta with duplex DNA and incoming nucleotide
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Lates calcarifer (barramundi perch)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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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 / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 1.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 825204 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0075382
ELECTRON MICROSCOPYf_angle_d0.7527385
ELECTRON MICROSCOPYf_dihedral_angle_d18.1072040
ELECTRON MICROSCOPYf_chiral_restr0.178847
ELECTRON MICROSCOPYf_plane_restr0.006867

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