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- EMDB-43874: Ternary complex of human DNA polymerase theta polymerase domain w... -

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Basic information

Entry
Database: EMDB / ID: EMD-43874
TitleTernary complex of human DNA polymerase theta polymerase domain with a mismatched T:T base pair
Map dataTernary complex of human DNA polymerase theta polymerase domain with a mismatched T:T base pair
Sample
  • Complex: Ternary complex of human DNA polymerase theta polymerase domain with a mismatched T:T base pair
    • Protein or peptide: DNA polymerase theta
    • DNA: DNA (5'-D(P*CP*GP*TP*AP*GP*(DOC))-3')
    • DNA: DNA (5'-D(P*AP*GP*TP*GP*CP*TP*AP*CP*G)-3')
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsDNA translesion synthesis / theta-mediated end joining / A-family DNA polymerase / DNA BINDING PROTEIN-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 polymerase theta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsLi C / Gao Y
Funding support United States, 2 items
OrganizationGrant numberCountry
American Cancer SocietyRSG-22-082-517 01-DMC United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR190046 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis of error-prone DNA synthesis by DNA polymerase θ.
Authors: Chuxuan Li / Leora M Maksoud / Yang Gao /
Abstract: DNA polymerase θ (Pol θ) is an A-family DNA polymerase specialized in DNA double-strand breaks repair and translesion synthesis. Distinct from its high-fidelity homologs in DNA replication, Pol θ ...DNA polymerase θ (Pol θ) is an A-family DNA polymerase specialized in DNA double-strand breaks repair and translesion synthesis. Distinct from its high-fidelity homologs in DNA replication, Pol θ catalyzes template-dependent DNA synthesis with an inherent propensity for error incorporation. However, the structural basis of Pol θ's low-fidelity DNA synthesis is not clear. Here, we present cryo-electron microscopy structures detailing the polymerase domain of human Pol θ in complex with a cognate C:G base pair (bp), a mismatched T:G bp, or a mismatched T:T bp. Our structures illustrate that Pol θ snugly accommodates the mismatched nascent base pairs within its active site with the finger domain well-closed, consistent with our in-solution fluorescence measurement but in contrast to its high-fidelity homologs. In addition, structural examination and mutagenesis study show that unique residues surrounding the active site contribute to the stabilization of the mismatched nascent base pair. Furthermore, Pol θ can efficiently extend from the misincorporated T:G or T:T mismatches, yet with a preference for template or primer looping-out, resulting in insertions and deletions. Collectively, our results elucidate how an A-family polymerase is adapted for error-prone DNA synthesis.
History
DepositionFeb 28, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43874.png

Downloads & links

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Map

FileDownload / File: emd_43874.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTernary complex of human DNA polymerase theta polymerase domain with a mismatched T:T base pair
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 256 pix.
= 209.92 Å		0.82 Å/pix.
x 256 pix.
= 209.92 Å		0.82 Å/pix.
x 256 pix.
= 209.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.469
Minimum - Maximum-3.2427838 - 4.2794685
Average (Standard dev.)0.000510735 (±0.088894814)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 209.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_43874_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_43874_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of human DNA polymerase theta polymerase domain w...

EntireName: Ternary complex of human DNA polymerase theta polymerase domain with a mismatched T:T base pair
Components
  • Complex: Ternary complex of human DNA polymerase theta polymerase domain with a mismatched T:T base pair
    • Protein or peptide: DNA polymerase theta
    • DNA: DNA (5'-D(P*CP*GP*TP*AP*GP*(DOC))-3')
    • DNA: DNA (5'-D(P*AP*GP*TP*GP*CP*TP*AP*CP*G)-3')
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of human DNA polymerase theta polymerase domain w...

SupramoleculeName: Ternary complex of human DNA polymerase theta polymerase domain with a mismatched T:T base pair
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA polymerase theta

MacromoleculeName: DNA polymerase theta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 89.585555 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: GFKDNSPISD TSFSLQLSQD GLQLTPASSS SESLSIIDVA SDQNLFQTFI KEWRCKKRFS ISLACEKIRS LTSSKTATIG SRFKQASSP QEIPIRDDGF PIKGCDDTLV VGLAVCWGGR DAYYFSLQKE QKHSEISASL VPPSLDPSLT LKDRMWYLQS C LRKESDKE ...String:
GFKDNSPISD TSFSLQLSQD GLQLTPASSS SESLSIIDVA SDQNLFQTFI KEWRCKKRFS ISLACEKIRS LTSSKTATIG SRFKQASSP QEIPIRDDGF PIKGCDDTLV VGLAVCWGGR DAYYFSLQKE QKHSEISASL VPPSLDPSLT LKDRMWYLQS C LRKESDKE CSVVIYDFIQ SYKILLLSCG ISLEQSYEDP KVACWLLDPD SQEPTLHSIV TSFLPHELPL LEGMETSQGI QS LGLNAGS EHSGRYRASV ESILIFNSMN QLNSLLQKEN LQDVFRKVEM PSQYCLALLE LNGIGFSTAE CESQKHIMQA KLD AIETQA YQLAGHSFSF TSSDDIAEVL FLELKLPPNR EMKNQGSKKT LGSTRRGIDN GRKLRLGRQF STSKDVLNKL KALH PLPGL ILEWRRITNA ITKVVFPLQR EKCLNPFLGM ERIYPVSQSH TATGRITFTE PNIQNVPRDF EIKMPTLVGE SPPSQ AVGK GLLPMGRGKY KKGFSVNPRC QAQMEERAAD RGMPFSISMR HAFVPFPGGS ILAADYSQLE LRILAHLSHD RRLIQV LNT GADVFRSIAA EWKMIEPESV GDDLRQQAKQ ICYGIIYGMG AKSLGEQMGI KENDAACYID SFKSRYTGIN QFMTETV KN CKRDGFVQTI LGRRRYLPGI KDNNPYRKAH AERQAINTIV QGSAADIVKI ATVNIQKQLE TFHSTFKSHG HREGMLQS D QTGLSRKRKL QGMFCPIRGG FFILQLHDEL LYEVAEEDVV QVAQIVKNEM ESAVKLSVKL KVKVKIGASW GELKDFDV

UniProtKB: DNA polymerase theta

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Macromolecule #2: DNA (5'-D(P*CP*GP*TP*AP*GP*(DOC))-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*TP*AP*GP*(DOC))-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.149978 KDa
SequenceString:
(DT)(DG)(DC)(DT)(DG)(DT)(DG)(DA)(DG)(DG) (DC)(DA)(DT)(DC)(DC)(DG)(DT)(DA)(DG) (DOC)

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

MacromoleculeName: DNA (5'-D(P*AP*GP*TP*GP*CP*TP*AP*CP*G)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.904736 KDa
SequenceString:
(DG)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DG) (DC)(DT)(DA)(DC)(DG)(DG)(DA)(DT)(DG)(DC) (DC)(DT)(DC)(DA)(DC)(DA)(DG)(DC)(DA)

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Macromolecule #4: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.7 mg/mL
BufferpH: 7.6
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 92385
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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