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- PDB-7lxd: Structure of yeast DNA Polymerase Zeta (apo) -

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Basic information

Entry
Database: PDB / ID: 7lxd
TitleStructure of yeast DNA Polymerase Zeta (apo)
Components
  • DNA polymerase delta small subunit
  • DNA polymerase delta subunit 3
  • DNA polymerase zeta catalytic subunit
  • DNA polymerase zeta processivity subunit
KeywordsDNA BINDING PROTEIN / nucleic acid binding / DNA polymerase / metal ion binding / catalytic activity
Function / homology
Function and homology information


delta DNA polymerase complex / H3-H4 histone complex chaperone activity / DNA amplification / zeta DNA polymerase complex / RNA-templated DNA biosynthetic process / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA replication, removal of RNA primer / Translesion synthesis by REV1 / Translesion synthesis by POLK ...delta DNA polymerase complex / H3-H4 histone complex chaperone activity / DNA amplification / zeta DNA polymerase complex / RNA-templated DNA biosynthetic process / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA replication, removal of RNA primer / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / lagging strand elongation / double-strand break repair via break-induced replication / postreplication repair / DNA metabolic process / DNA strand elongation involved in DNA replication / error-free translesion synthesis / leading strand elongation / mismatch repair / error-prone translesion synthesis / nucleotide-excision repair / double-strand break repair via homologous recombination / base-excision repair / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin / mitochondrion / DNA binding / nucleus / metal ion binding / cytosol
Similarity search - Function
DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase zeta catalytic subunit / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / Mad2-like / HORMA domain / HORMA domain ...DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase zeta catalytic subunit / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA polymerase zeta catalytic subunit / DNA polymerase zeta processivity subunit / DNA polymerase delta small subunit / DNA polymerase delta subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.11 Å
AuthorsTruong, C.D. / Craig, T.A. / Cui, G. / Botuyan, M.V. / Serkasevich, R.A. / Chan, K.-Y. / Mer, G. / Chiu, P.-L. / Kumar, R.
CitationJournal: J Biol Chem / Year: 2021
Title: Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ.
Authors: Chloe Du Truong / Theodore A Craig / Gaofeng Cui / Maria Victoria Botuyan / Rachel A Serkasevich / Ka-Yi Chan / Georges Mer / Po-Lin Chiu / Rajiv Kumar /
Abstract: The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. ...The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.
History
DepositionMar 3, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 30, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • EMDB-23570
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Assembly

Deposited unit
A: DNA polymerase zeta catalytic subunit
D: DNA polymerase zeta processivity subunit
E: DNA polymerase zeta processivity subunit
F: DNA polymerase delta small subunit
G: DNA polymerase delta subunit 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)327,1146
Polymers326,7635
Non-polymers3521
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, Negative stain EM
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DNA polymerase zeta catalytic subunit / Protein reversionless 3


Mass: 173197.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Cell line: Saccharomyces cerevisiae / Gene: REV3, PSO1, YPL167C, P2535 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): PY265
Variant (production host): can 1 his3 leu 2 trp 1 ura 3 pep4:HIS3 GAL nam7delta::Mx4
References: UniProt: P14284, DNA-directed DNA polymerase
#2: Protein DNA polymerase zeta processivity subunit / Revertibility protein 7


Mass: 28791.654 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: REV7, YIL139C / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): PY265
Variant (production host): can 1 his3 leu 2 trp 1 ura 3 pep4:HIS3 GAL nam7delta::Mx4
References: UniProt: P38927
#3: Protein DNA polymerase delta small subunit / Hydroxyurea-sensitive protein 2


Mass: 55603.992 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: POL31, HUS2, HYS2, SDP5, YJR006W, J1427, YJR83.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): PY265
Variant (production host): can 1 his3 leu 2 trp 1 ura 3 pep4:HIS3 GAL nam7delta::Mx4
References: UniProt: P46957, DNA-directed DNA polymerase
#4: Protein DNA polymerase delta subunit 3


Mass: 40377.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: POL32, YJR043C, J1626 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): PY265
Variant (production host): can 1 his3 leu 2 trp 1 ura 3 pep4:HIS3 GAL nam7delta::Mx4
References: UniProt: P47110
#5: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
Has ligand of interestN

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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: DNA polymerase Zeta / Type: COMPLEX
Details: DNA polymerase Zeta is generated from yeast without DNA binding.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.326 MDaNO
21172 kDa/nmYES
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: PY265 / Cell: Yeast / Plasmid: pBL813, pBL347, pBL824
Buffer solutionpH: 6.9
Details: Solutions were made fresh from concentrate to avoid microbial contamination. They are further filtered using 0.2 micrometer filtering membrane and a pressure/vacuum filtration unit.
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM2-(N-morpholino)ethanesulfonic acidMES1
2120 mMSodium ChlorideNaCl1
35 mMCalcium ChlorideCaCl21
42 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono disperse.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: Blot for 6 seconds before plunging

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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 FIELD / Calibrated magnification: 48780 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 45.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11698
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEM3.9image acquisition
4cryoSPARC3CTF correctionPatch CTF estimation was used to determine the CTF correction
7UCSF Chimera1.14model fitting
9PHENIX1.18.2-3874model refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
12cryoSPARC3classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2974553
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213120 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 6V8P

6v8p


Accession code: 6V8P / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00617855
ELECTRON MICROSCOPYf_angle_d1.29924162
ELECTRON MICROSCOPYf_dihedral_angle_d19.5396708
ELECTRON MICROSCOPYf_chiral_restr0.0742691
ELECTRON MICROSCOPYf_plane_restr0.0093064

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