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- PDB-9bp9: Human DNA polymerase theta helicase domain in complex with inhibi... -

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Basic information

Entry
Database: PDB / ID: 9bp9
TitleHuman DNA polymerase theta helicase domain in complex with inhibitor AB25583, dimer form
ComponentsDNA polymerase theta
KeywordsTRANSFERASE/INHIBITOR / DNA repair / helicase / ATPase / TRANSFERASE-INHIBITOR 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 / 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 / mitochondrial nucleoid / site of DNA damage / 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: 3.21 Å
AuthorsIto, F. / Li, Z. / Chen, X.S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM130889 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM.
Authors: Fumiaki Ito / Ziyuan Li / Leonid Minakhin / Gurushankar Chandramouly / Mrityunjay Tyagi / Robert Betsch / John J Krais / Bernadette Taberi / Umeshkumar Vekariya / Marissa Calbert / Tomasz ...Authors: Fumiaki Ito / Ziyuan Li / Leonid Minakhin / Gurushankar Chandramouly / Mrityunjay Tyagi / Robert Betsch / John J Krais / Bernadette Taberi / Umeshkumar Vekariya / Marissa Calbert / Tomasz Skorski / Neil Johnson / Xiaojiang S Chen / Richard T Pomerantz /
Abstract: DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision ...DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.
History
DepositionMay 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA polymerase theta
C: DNA polymerase theta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)200,5114
Polymers199,6052
Non-polymers9062
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: Protein DNA polymerase theta / DNA polymerase eta


Mass: 99802.539 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-directed DNA polymerase
#2: Chemical ChemComp-WCN / (4P)-N-{5-[(4-chlorophenyl)methoxy]-1,3,4-thiadiazol-2-yl}-4-(2-methoxyphenyl)pyridine-3-carboxamide


Mass: 452.913 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H17ClN4O3S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 complex with inhibitor AB25583
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.199 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 0.8 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

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2 mm / C2 aperture diameter: 50 µm
Image recordingAverage exposure time: 8 sec. / Electron dose: 58 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4500

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Processing

EM softwareName: EPU / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2932534
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 673324 / Symmetry type: POINT

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