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- EMDB-43818: Human DNA polymerase theta helicase domain tetramer, apo-form -

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Basic information

Entry
Database: EMDB / ID: EMD-43818
TitleHuman DNA polymerase theta helicase domain tetramer, apo-form
Map data
Sample
  • Complex: Human DNA polymerase theta helicase domain tetramer, apo-form
    • Protein or peptide: DNA polymerase theta
KeywordsDNA repair / helicase / ATPase / TRANSFERASE / DNA BINDING PROTEIN
Function / homology
Function and homology information


single-stranded DNA endodeoxyribonuclease activity / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / double-strand break repair via alternative nonhomologous end joining / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / negative regulation of double-strand break repair via homologous recombination / error-prone translesion synthesis ...single-stranded DNA endodeoxyribonuclease activity / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / double-strand break repair via alternative nonhomologous end joining / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / negative regulation of double-strand break repair via homologous recombination / 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 polymerase theta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsIto F / Li Z / Chen XS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM130889 United States
CitationJournal: 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
DepositionFeb 26, 2024-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43818.png

Downloads & links

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Map

FileDownload / File: emd_43818.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 400 pix.
= 368. Å		0.92 Å/pix.
x 400 pix.
= 368. Å		0.92 Å/pix.
x 400 pix.
= 368. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.56369054 - 0.9252917
Average (Standard dev.)-0.000020853948 (±0.01488616)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 368.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43818_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_43818_half_map_2.map
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Sample components

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Entire : Human DNA polymerase theta helicase domain tetramer, apo-form

EntireName: Human DNA polymerase theta helicase domain tetramer, apo-form
Components
  • Complex: Human DNA polymerase theta helicase domain tetramer, apo-form
    • Protein or peptide: DNA polymerase theta

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Supramolecule #1: Human DNA polymerase theta helicase domain tetramer, apo-form

SupramoleculeName: Human DNA polymerase theta helicase domain tetramer, apo-form
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 398 KDa

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Macromolecule #1: DNA polymerase theta

MacromoleculeName: DNA polymerase theta / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.802539 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNLLRRSGKR RRSESGSDSF SGSGGDSSAS PQFLSGSVLS PPPGLGRCLK AAAAGECKPT VPDYERDKLL LANWGLPKAV LEKYHSFGV KKMFEWQAEC LLLGQVLEGK NLVYSAPTSA GKTLVAELLI LKRVLEMRKK ALFILPFVSV AKEKKYYLQS L FQEVGIKV ...String:
MNLLRRSGKR RRSESGSDSF SGSGGDSSAS PQFLSGSVLS PPPGLGRCLK AAAAGECKPT VPDYERDKLL LANWGLPKAV LEKYHSFGV KKMFEWQAEC LLLGQVLEGK NLVYSAPTSA GKTLVAELLI LKRVLEMRKK ALFILPFVSV AKEKKYYLQS L FQEVGIKV DGYMGSTSPS RHFSSLDIAV CTIERANGLI NRLIEENKMD LLGMVVVDEL HMLGDSHRGY LLELLLTKIC YI TRKSASC QADLASSLSN AVQIVGMSAT LPNLELVASW LNAELYHTDF RPVPLLESVK VGNSIYDSSM KLVREFEPML QVK GDEDHV VSLCYETICD NHSVLLFCPS KKWCEKLADI IAREFYNLHH QAEGLVKPSE CPPVILEQKE LLEVMDQLRR LPSG LDSVL QKTVPWGVAF HHAGLTFEER DIIEGAFRQG LIRVLAATST LSSGVNLPAR RVIIRTPIFG GRPLDILTYK QMVGR AGRK GVDTVGESIL ICKNSEKSKG IALLQGSLKP VRSCLQRREG EEVTGSMIRA ILEIIVGGVA STSQDMHTYA ACTFLA ASM KEGKQGIQRN QESVQLGAIE ACVMWLLENE FIQSTEASDG TEGKVYHPTH LGSATLSSSL SPADTLDIFA DLQRAMK GF VLENDLHILY LVTPMFEDWT TIDWYRFFCL WEKLPTSMKR VAELVGVEEG FLARCVKGKV VARTERQHRQ MAIHKRFF T SLVLLDLISE VPLREINQKY GCNRGQIQSL QQSAAVYAGM ITVFSNRLGW HNMELLLSQF QKRLTFGIQR ELCDLVRVS LLNAQRARVL YASGFHTVAD LARANIVEVE VILKNAVPFK SARKAVDEEE EAVEERRNMR TIWVTGRKGL TEREAAALIV EEARMILQQ DLVEM

UniProtKB: DNA polymerase theta

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4511 / Average exposure time: 8.0 sec. / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 150000

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Image processing

Particle selectionNumber selected: 3961362
Startup modelType of model: INSILICO MODEL
In silico model: Generated by stochastic gradient descent using ab initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 202004
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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