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- EMDB-23299: The negative stain EM structure of the DNA Ligase III catalytic c... -

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Basic information

Entry
Database: EMDB / ID: EMD-23299
TitleThe negative stain EM structure of the DNA Ligase III catalytic core in complex with TDP1.
Map dataDNA LigIII catalytic fragment in complex with TDP1
Sample
  • Complex: DNA LigIII catalytic core in complex with TDP1
    • Protein or peptide: Nuclear DNA ligase III
    • Protein or peptide: Tyrosyl-DNA phosphodiesterase 1
KeywordsDNA repair / protein-protein interactions / DNA BINDING PROTEIN
Function / homology
Function and homology information


DNA ligase III-XRCC1 complex / negative regulation of mitochondrial DNA replication / 3'-tyrosyl-DNA phosphodiesterase activity / base-excision repair, DNA ligation / DNA ligase activity / DNA ligase (ATP) / DNA ligase (ATP) activity / single strand break repair / Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases / DNA ligation ...DNA ligase III-XRCC1 complex / negative regulation of mitochondrial DNA replication / 3'-tyrosyl-DNA phosphodiesterase activity / base-excision repair, DNA ligation / DNA ligase activity / DNA ligase (ATP) / DNA ligase (ATP) activity / single strand break repair / Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases / DNA ligation / HDR through MMEJ (alt-NHEJ) / lagging strand elongation / exonuclease activity / mitochondrial DNA repair / Resolution of AP sites via the single-nucleotide replacement pathway / DNA biosynthetic process / double-strand break repair via alternative nonhomologous end joining / APEX1-Independent Resolution of AP Sites via the Single Nucleotide Replacement Pathway / mitochondrion organization / Gap-filling DNA repair synthesis and ligation in GG-NER / base-excision repair, gap-filling / Nonhomologous End-Joining (NHEJ) / double-strand break repair via homologous recombination / Gap-filling DNA repair synthesis and ligation in TC-NER / double-strand break repair / single-stranded DNA binding / double-stranded DNA binding / cell cycle / cell division / intracellular membrane-bounded organelle / DNA repair / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
DNA ligase 3, BRCT domain / DNA ligase 3 BRCT domain / Tyrosyl-DNA phosphodiesterase I / Tyrosyl-DNA phosphodiesterase / DNA ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / DNA ligase N terminus / ATP-dependent DNA ligase signature 2. / ATP-dependent DNA ligase AMP-binding site. ...DNA ligase 3, BRCT domain / DNA ligase 3 BRCT domain / Tyrosyl-DNA phosphodiesterase I / Tyrosyl-DNA phosphodiesterase / DNA ligase, ATP-dependent / DNA ligase, ATP-dependent, N-terminal / DNA ligase, ATP-dependent, N-terminal domain superfamily / DNA ligase N terminus / ATP-dependent DNA ligase signature 2. / ATP-dependent DNA ligase AMP-binding site. / DNA ligase, ATP-dependent, C-terminal / ATP dependent DNA ligase C terminal region / DNA ligase, ATP-dependent, conserved site / ATP-dependent DNA ligase family profile. / Zinc finger poly(ADP-ribose) polymerase (PARP)-type signature. / Zinc finger, PARP-type superfamily / Poly(ADP-ribose) polymerase and DNA-Ligase Zn-finger region / Zinc finger poly(ADP-ribose) polymerase (PARP)-type profile. / Poly(ADP-ribose) polymerase and DNA-Ligase Zn-finger region / DNA ligase, ATP-dependent, central / ATP dependent DNA ligase domain / Zinc finger, PARP-type / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA ligase 3 / Tyrosyl-DNA phosphodiesterase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 28.0 Å
AuthorsSverzhinsky A / Pascal JM
Funding support United States, Canada, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 ES012512 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 CA22043 United States
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2015-05776 Canada
Other governmentP01 CA92584 United States
CitationJournal: J Biol Chem / Year: 2021
Title: Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus.
Authors: Ishtiaque Rashid / Michal Hammel / Aleksandr Sverzhinsky / Miaw-Sheue Tsai / John M Pascal / John A Tainer / Alan E Tomkinson /
Abstract: Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA ...Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.
History
DepositionJan 15, 2021-
Header (metadata) releaseJul 14, 2021-
Map releaseJul 14, 2021-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0381
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0381
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23299.png

Downloads & links

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Map

FileDownload / File: emd_23299.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDNA LigIII catalytic fragment in complex with TDP1
Voxel sizeX=Y=Z: 3.3 Å
Density
Contour LevelBy AUTHOR: 0.0381 / Movie #1: 0.0381
Minimum - Maximum-0.06105871 - 0.11781523
Average (Standard dev.)0.0021912656 (±0.013781659)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.33.33.3
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z211.200211.200211.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-0.0610.1180.002

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Supplemental data

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Sample components

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Entire : DNA LigIII catalytic core in complex with TDP1

EntireName: DNA LigIII catalytic core in complex with TDP1
Components
  • Complex: DNA LigIII catalytic core in complex with TDP1
    • Protein or peptide: Nuclear DNA ligase III
    • Protein or peptide: Tyrosyl-DNA phosphodiesterase 1

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Supramolecule #1: DNA LigIII catalytic core in complex with TDP1

SupramoleculeName: DNA LigIII catalytic core in complex with TDP1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 124 KDa

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Macromolecule #1: Nuclear DNA ligase III

MacromoleculeName: Nuclear DNA ligase III / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: DNA ligase (ATP)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHSSG LVPRGSHMRH KDCLLREFRK LCAMVADNPS YNTKTQIIQD FLRKGSAGDG FHGDVYLTVK LLLPGVIKTV YNLNDKQIVK LFSRIFNCNP DDMARDLEQG DVSETIRVFF EQSKSFPPAA KSLLTIQEVD EFLLRLSKLT KEDEQQQALQ DIASRCTAND ...String:
MHHHHHHSSG LVPRGSHMRH KDCLLREFRK LCAMVADNPS YNTKTQIIQD FLRKGSAGDG FHGDVYLTVK LLLPGVIKTV YNLNDKQIVK LFSRIFNCNP DDMARDLEQG DVSETIRVFF EQSKSFPPAA KSLLTIQEVD EFLLRLSKLT KEDEQQQALQ DIASRCTAND LKCIIRLIKH DLKMNSGAKH VLDALDPNAY EAFKASRNLQ DVVERVLHNA QEVEKEPGQR RALSVQASLM TPVQPMLAEA CKSVEYAMKK CPNGMFSEIK YDGERVQVHK NGDHFSYFSR SLKPVLPHKV AHFKDYIPQA FPGGHSMILD SEVLLIDNKT GKPLPFGTLG VHKKAAFQDA NVCLFVFDCI YFNDVSLMDR PLCERRKFLH DNMVEIPNRI MFSEMKRVTK ALDLADMITR VIQEGLEGLV LKDVKGTYEP GKRHWLKVKK DYLNEGAMAD TADLVVLGAF YGQGSKGGMM SIFLMGCYDP GSQKWCTVTK CAGGHDDATL ARLQNELDMV KISKDPSKIP SWLKVNKIYY PDFIVPDPKK AAVWEITGAE FSKSEAHTAD GISIRFPRCT RIRDDKDWKS ATNLPQLKEL YQLSKEKADF TVVA

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Macromolecule #2: Tyrosyl-DNA phosphodiesterase 1

MacromoleculeName: Tyrosyl-DNA phosphodiesterase 1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDYKDDDDKE FTMSQEGDYG RWTISSSDES EEEKPKPDKP STSSLLCARQ GAANEPRYTC SEAQKAAHKR KISPVKFSNT DSVLPPKRQK SGSQEDLGWC LSSSDDELQP EMPQKQAEKV VIKKEKDISA PNDGTAQRTE NHGAPACHRL KEEEDEYETS GEGQDIWDML ...String:
MDYKDDDDKE FTMSQEGDYG RWTISSSDES EEEKPKPDKP STSSLLCARQ GAANEPRYTC SEAQKAAHKR KISPVKFSNT DSVLPPKRQK SGSQEDLGWC LSSSDDELQP EMPQKQAEKV VIKKEKDISA PNDGTAQRTE NHGAPACHRL KEEEDEYETS GEGQDIWDML DKGNPFQFYL TRVSGVKPKY NSGALHIKDI LSPLFGTLVS SAQFNYCFDV DWLVKQYPPE FRKKPILLVH GDKREAKAHL HAQAKPYENI SLCQAKLDIA FGTHHTKMML LLYEEGLRVV IHTSNLIHAD WHQKTQGIWL SPLYPRIADG THKSGESPTH FKADLISYLM AYNAPSLKEW IDVIHKHDLS ETNVYLIGST PGRFQGSQKD NWGHFRLKKL LKDHASSMPN AESWPVVGQF SSVGSLGADE SKWLCSEFKE SMLTLGKESK TPGKSSVPLY LIYPSVENVR TSLEGYPAGG SLPYSIQTAE KQNWLHSYFH KWSAETSGRS NAMPHIKTYM RPSPDFSKIA WFLVTSANLS KAAWGALEKN GTQLMIRSYE LGVLFLPSAF GLDSFKVKQK FFAGSQEPMA TFPVPYDLPP ELYGSKDRPW IWNIPYVKAP DTHGNMWVPS

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.0025 mg/mL
BufferpH: 7.5
StainingType: NEGATIVE / Material: Uranyl Formate
GridMaterial: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
DetailsCo-purified proteins were crosslinked with glutaraldehyde, then buffer exchanged to remove the crosslinker before negative staining.

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Average exposure time: 1.0 sec. / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 67000

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Image processing

Particle selectionNumber selected: 22300
Startup modelType of model: OTHER / Details: SGD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 10430
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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