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- PDB-9bh7: Human DNA polymerase theta helicase domain dimer in the apo form -

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Basic information

Entry
Database: PDB / ID: 9bh7
TitleHuman DNA polymerase theta helicase domain dimer in the apo form
ComponentsDNA polymerase theta
KeywordsDNA BINDING PROTEIN / DNA repair / TMEJ / MMEJ
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
DNA polymerase theta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZerio, C.J. / Lander, G.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)F32CA288144 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10OD032467 United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Human polymerase θ helicase positions DNA microhomologies for double-strand break repair.
Authors: Christopher J Zerio / Yonghong Bai / Brian A Sosa-Alvarado / Timothy Guzi / Gabriel C Lander /
Abstract: DNA double-strand breaks occur daily in all human cells and must be repaired with high fidelity to minimize genomic instability. Deficiencies in high-fidelity DNA repair by homologous recombination ...DNA double-strand breaks occur daily in all human cells and must be repaired with high fidelity to minimize genomic instability. Deficiencies in high-fidelity DNA repair by homologous recombination lead to dependence on DNA polymerase θ, which identifies DNA microhomologies in 3' single-stranded DNA overhangs and anneals them to initiate error-prone double-strand break repair. The resulting genomic instability is associated with numerous cancers, thereby making this polymerase an attractive therapeutic target. However, despite the biomedical importance of polymerase θ, the molecular details of how it initiates DNA break repair remain unclear. Here, we present cryo-electron microscopy structures of the polymerase θ helicase domain bound to microhomology-containing DNA, revealing DNA-induced rearrangements of the helicase that enable DNA repair. Our structures show that DNA-bound helicase dimers facilitate a microhomology search that positions 3' single-stranded DNA ends in proximity to align complementary bases and anneal DNA microhomology. We characterize the molecular determinants that enable the helicase domain of polymerase θ to identify and pair DNA microhomologies to initiate mutagenic DNA repair, thereby providing insight into potentially targetable interactions for therapeutic interventions.
History
DepositionApr 19, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA polymerase theta
B: DNA polymerase theta


Theoretical massNumber of molelcules
Total (without water)199,3432
Polymers199,3432
Non-polymers00
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: 99671.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLQ, POLH / Plasmid: pET-Duet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta
References: UniProt: O75417, DNA helicase, DNA-directed DNA polymerase, RNA-directed DNA polymerase
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: Human DNA polymerase theta helicase domain / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta(DE3) / Plasmid: pET-Duet-1
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHepes-NaOH1
2250 mMSodium chlorideNaCl1
30.5 mMTCEP1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow discharged under vacuum for 30 s at 15 mA in a Pelco easiGlow 91000 Glow Discharge Cleaning System.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: 3 microliters of sample was applied to the surface of the grid, blotted with Whatman 1 filter paper until 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid ...Details: 3 microliters of sample was applied to the surface of the grid, blotted with Whatman 1 filter paper until 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid was plunged into a liquid ethane pool cooled by liquid nitrogen using a manual plunge freezer.

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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 magnification: 105000 X / Calibrated magnification: 60024 X / Nominal defocus max: 1000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 150 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 70 K
Image recordingAverage exposure time: 0.9 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3906
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.3particle selectionTemplate picker
2Leginon3.6image acquisition
4cryoSPARC4.3CTF correctionLive Patch CTF
7UCSF ChimeraX1.6.1model fitting
8ISOLDE1.6.0model fittingflexible local modeling
10PHENIX1.20.1model refinementrigid real-space refinement
11cryoSPARC4.3initial Euler assignment
12cryoSPARC4.3final Euler assignment
13cryoSPARC4.3classification
14cryoSPARC4.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1474207
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138712 / Algorithm: FOURIER SPACE
Details: We performed local refinement of a single protomer, and then multiplied the output protomer map to fit the C2 symmetric dimer map.
Symmetry type: POINT
Atomic model buildingB value: 55.33 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: We used ISOLDE to flexibly fit one protomer into the locally refined protomer map. The output protomer model was multiplied and we used ISOLDE to fit two protomers into the dimer map.
Atomic model buildingPDB-ID: 5A9J

5a9j


Pdb chain-ID: A / Accession code: 5A9J / Chain residue range: 67-892 / Pdb chain residue range: 67-892 / Source name: PDB / Type: experimental model

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