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- PDB-9au9: Ternary complex of human DNA polymerase theta polymerase domain w... -

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Basic information

Entry
Database: PDB / ID: 9au9
TitleTernary complex of human DNA polymerase theta polymerase domain with a mismatched T:G base pair
Components
  • DNA (5'-D(P*GP*TP*GP*CP*TP*AP*CP*GP*GP*AP*TP*GP*CP*CP*TP*CP*A)-3')
  • DNA (5'-D(P*TP*GP*AP*GP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(DOC))-3')
  • DNA polymerase theta
KeywordsDNA BINDING PROTEIN/DNA / DNA translesion synthesis / theta-mediated end joining / A-family DNA polymerase / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / replication fork processing / site of DNA damage / mitochondrial nucleoid / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / error-prone translesion synthesis / negative regulation of double-strand break repair via homologous recombination ...double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / replication fork processing / site of DNA damage / mitochondrial nucleoid / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / error-prone translesion synthesis / negative regulation of double-strand break repair via homologous recombination / RNA-directed DNA polymerase activity / DNA helicase activity / base-excision repair / protein homooligomerization / RNA-directed DNA polymerase / double-strand break repair / site of double-strand break / DNA helicase / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / DNA damage response / chromatin binding / magnesium ion binding / Golgi apparatus / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol
Similarity search - Function
: / DNA_pol_Q helicase like region helical domain / DNA polymerase theta-like, helix-turn-helix domain / Helix-turn-helix domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain ...: / DNA_pol_Q helicase like region helical domain / DNA polymerase theta-like, helix-turn-helix domain / Helix-turn-helix domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA polymerase theta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsLi, C. / Gao, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
American Cancer SocietyRSG-22-082-517 01-DMC United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR190046 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis of error-prone DNA synthesis by DNA polymerase θ.
Authors: Chuxuan Li / Leora M Maksoud / Yang Gao /
Abstract: DNA polymerase θ (Pol θ) is an A-family DNA polymerase specialized in DNA double-strand breaks repair and translesion synthesis. Distinct from its high-fidelity homologs in DNA replication, Pol θ ...DNA polymerase θ (Pol θ) is an A-family DNA polymerase specialized in DNA double-strand breaks repair and translesion synthesis. Distinct from its high-fidelity homologs in DNA replication, Pol θ catalyzes template-dependent DNA synthesis with an inherent propensity for error incorporation. However, the structural basis of Pol θ's low-fidelity DNA synthesis is not clear. Here, we present cryo-electron microscopy structures detailing the polymerase domain of human Pol θ in complex with a cognate C:G base pair (bp), a mismatched T:G bp, or a mismatched T:T bp. Our structures illustrate that Pol θ snugly accommodates the mismatched nascent base pairs within its active site with the finger domain well-closed, consistent with our in-solution fluorescence measurement but in contrast to its high-fidelity homologs. In addition, structural examination and mutagenesis study show that unique residues surrounding the active site contribute to the stabilization of the mismatched nascent base pair. Furthermore, Pol θ can efficiently extend from the misincorporated T:G or T:T mismatches, yet with a preference for template or primer looping-out, resulting in insertions and deletions. Collectively, our results elucidate how an A-family polymerase is adapted for error-prone DNA synthesis.
History
DepositionFeb 28, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 28, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase theta
C: DNA (5'-D(P*TP*GP*AP*GP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(DOC))-3')
D: DNA (5'-D(P*GP*TP*GP*CP*TP*AP*CP*GP*GP*AP*TP*GP*CP*CP*TP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,1474
Polymers104,6403
Non-polymers5071
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein DNA polymerase theta / DNA polymerase eta


Mass: 89585.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLQ, POLH
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: O75417, DNA-directed DNA polymerase
#2: DNA chain DNA (5'-D(P*TP*GP*AP*GP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(DOC))-3')


Mass: 6149.978 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA (5'-D(P*GP*TP*GP*CP*TP*AP*CP*GP*GP*AP*TP*GP*CP*CP*TP*CP*A)-3')


Mass: 8904.736 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#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
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 human DNA polymerase theta with duplex DNA and incoming nucleotide
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.6
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 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

EM softwareName: PHENIX / Version: 1.21_5207: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93767 / Symmetry type: POINT

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