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- PDB-9c5q: Human DNA polymerase theta helicase domain in microhomology annea... -

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Basic information

Entry
Database: PDB / ID: 9c5q
TitleHuman DNA polymerase theta helicase domain in microhomology annealed state 2, dimer form
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*CP*CP*CP*GP*GP*G)-3')
  • DNA polymerase theta
KeywordsTRANSFERASE/DNA / DNA repair / helicase / ATPase / TRANSFERASE-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 / mitochondrial nucleoid / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / error-prone translesion synthesis / negative regulation of double-strand break repair via homologous recombination / somatic hypermutation of immunoglobulin genes ...double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / replication fork processing / mitochondrial nucleoid / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / error-prone translesion synthesis / negative regulation of double-strand break repair via homologous recombination / somatic hypermutation of immunoglobulin genes / DNA helicase activity / base-excision repair / protein homooligomerization / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / double-strand break repair / site of double-strand break / DNA helicase / DNA-directed DNA polymerase / damaged DNA binding / 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 / Domain of unknown function (DUF7898) / 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_pol_Q helicase like region helical domain / Domain of unknown function (DUF7898) / 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 / DNA (> 10) / DNA polymerase theta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsIto, F. / Li, Z. / Chen, X.S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM152198 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI150524 United States
Citation
Journal: Nat Commun / Year: 2025
Title: Structural basis for Polθ-helicase DNA binding and microhomology-mediated end-joining.
Authors: Fumiaki Ito / Ziyuan Li / Leonid Minakhin / Htet A Khant / Richard T Pomerantz / Xiaojiang S Chen /
Abstract: DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous ...DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing different 3'-ssDNA overhangs. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-ssDNA overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel.
#1: Journal: bioRxiv / Year: 2024
Title: Structural Basis for Polθ-Helicase DNA Binding and Microhomology-Mediated End-Joining.
Authors: Fumiaki Ito / Ziyuan Li / Leonid Minakhin / Htet A Khant / Richard T Pomerantz / Xiaojiang S Chen /
Abstract: DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous ...DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel.
History
DepositionJun 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update
Revision 1.2Apr 30, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*CP*CP*CP*GP*GP*G)-3')
C: DNA polymerase theta
B: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*CP*CP*CP*GP*GP*G)-3')
A: DNA polymerase theta


Theoretical massNumber of molelcules
Total (without water)192,6674
Polymers192,6674
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*CP*CP*CP*GP*GP*G)-3')


Mass: 4243.748 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: Protein DNA polymerase theta / DNA polymerase eta


Mass: 92089.797 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLQ, POLH / Production host: Escherichia coli (E. coli)
References: UniProt: O75417, DNA helicase, DNA-directed DNA polymerase, RNA-directed DNA polymerase
Has protein modificationY

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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 in microhomology annealed state 2, dimer form
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.253 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingAverage exposure time: 3.5 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10005

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Processing

EM softwareName: EPU / Version: 3.5.0 / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3203827
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78928 / Num. of class averages: 1 / Symmetry type: POINT

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