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- PDB-9bh9: Human DNA polymerase theta helicase domain dimer bound to DNA in ... -

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Basic information

Entry
Database: PDB / ID: 9bh9
TitleHuman DNA polymerase theta helicase domain dimer bound to DNA in the microhomology aligning conformation
Components
  • DNA polymerase thetaPOLQ
  • Stem-loop DNA with microhomology in the 3' overhang
KeywordsDNA BINDING PROTEIN / DNA repair / TMEJ / MMEJ
Function / homology
Function and homology information


single-stranded DNA endodeoxyribonuclease activity / double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / negative regulation of double-strand break repair via homologous recombination / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / error-prone translesion synthesis ...single-stranded DNA endodeoxyribonuclease activity / double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / negative regulation of double-strand break repair via homologous recombination / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / error-prone translesion synthesis / 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 / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / DNA damage response / chromatin binding / Golgi apparatus / magnesium ion binding / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA polymerase theta-like, helix-turn-helix domain / : / Helix-turn-helix domain / DNA_pol_Q helicase like region helical 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 polymerase theta-like, helix-turn-helix domain / : / Helix-turn-helix domain / DNA_pol_Q helicase like region helical 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 / DEAD/DEAH box helicase domain / 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)
synthetic construct (others)
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: To Be Published
Title: Human polymerase theta helicase positions DNA microhomologies for double-strand break repair
Authors: Zerio, C.J. / Bai, Y. / Sosa-Alvarado, B.A. / Guzi, T. / Lander, G.C.
History
DepositionApr 19, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase theta
B: DNA polymerase theta
X: Stem-loop DNA with microhomology in the 3' overhang
Y: Stem-loop DNA with microhomology in the 3' overhang


Theoretical massNumber of molelcules
Total (without water)233,8714
Polymers233,8714
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, native gel electrophoresis, We used native PAGE to confirm protein binding to DNA
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein DNA polymerase theta / POLQ / 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
#2: DNA chain Stem-loop DNA with microhomology in the 3' overhang


Mass: 17264.012 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human DNA polymerase theta helicase domain bound to stem-loop DNA with microhomology in the 3' overhangCOMPLEXall0RECOMBINANT
2Human DNA polymerase theta helicase domainCOMPLEX#11RECOMBINANT
3Stem-loop DNA with microhomology in the 3' overhangCOMPLEX#21NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.22 MDaNO
210.2 MDaNO
310.02 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli (E. coli)562Rosetta(DE3)pET-Duet-1
32Escherichia coli (E. coli)562Rosetta(DE3)pET-Duet-1
43Escherichia coli (E. coli)562Rosetta(DE3)pET-Duet-1
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHepes-NaOH1
2100 mMSodium chlorideNaClSodium chloride1
30.5 mMTCEP1
SpecimenConc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: We added 2X pre-annealed DNA to the protein, lowered the NaCl concentration to 100 mM, and purified the DNA-bound complex by SEC.
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 for 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid was plunged into a liquid ...Details: 3 microliters of sample was applied to the surface of the grid, blotted for 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid was plunged into a liquid ethane bath cooled by liquid nitrogen.

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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 FIELDBright-field microscopy / 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: 2500 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: 1.4 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: 12440
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.3particle selectionLive template picker
2Leginon3.6image acquisition
4cryoSPARC4.3CTF correctionLive Patch CTF
7UCSF ChimeraX1.6.1model fitting
8ISOLDE1.6.0model fittingflexible local modeling
10cryoSPARC4.3initial Euler assignment
11cryoSPARC4.3final Euler assignment
12cryoSPARC4.3classification
13cryoSPARC4.33D reconstruction
14PHENIX1.20.1model refinementrigid real-space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5424691
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107353 / Algorithm: FOURIER SPACE
Details: Non-uniform refinement and local filtering by resolution
Symmetry type: POINT
Atomic model buildingB value: 67.19 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: We modeled DNA bases into sharpened density when possible. We modeled the DNA backbone into locally filtered density in low resolution areas.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeDetailsInitial refinement model-IDPdb chain residue range
15A9J

5a9j

A5A9JA67-892Protein167-892
22P6R

2p6r

X2P6RX6-19DNA26-19

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