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- PDB-8fok: Cryo-EM structure of S. cerevisiae DNA polymerase alpha-primase c... -

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Basic information

Entry
Database: PDB / ID: 8fok
TitleCryo-EM structure of S. cerevisiae DNA polymerase alpha-primase complex in the DNA elongation state
Components
  • DNA polymerase
  • DNA polymerase alpha subunit B
  • DNA primase
  • DNA primase large subunit
  • RNA-DNA chimeric primer
  • template DNA
KeywordsDNA BINDING PROTEIN / DNA polymerase / primase / DNA replication
Function / homology
Function and homology information


alpha DNA polymerase:primase complex / DNA primase activity / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / leading strand elongation / DNA replication origin binding / DNA replication initiation / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / single-stranded DNA binding ...alpha DNA polymerase:primase complex / DNA primase activity / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / leading strand elongation / DNA replication origin binding / DNA replication initiation / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / DNA binding / metal ion binding
Similarity search - Function
DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily ...DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / 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 / DNA (> 10) / RNA / RNA (> 10) / DNA primase large subunit / DNA polymerase / DNA primase / DNA polymerase alpha subunit B
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsYuan, Z. / Georgescu, R. / Li, H. / O'Donnell, M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115809 United States
Howard Hughes Medical Institute (HHMI)M.E.O. United States
Citation
Journal: Nat Commun / Year: 2023
Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase.
Authors: Zuanning Yuan / Roxana Georgescu / Huilin Li / Michael E O'Donnell /
Abstract: The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA ...The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively. It has been unclear how Pol α hands over an RNA primer from Pri1 to Pol1 for DNA primer extension, and how the primer length is defined. Here we report the cryo-EM analysis of yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states, revealing a series of very large movements. We reveal a critical point at which Pol1-core moves to take over the 3'-end of the RNA from Pri1. DNA extension is limited by a spiral motion of Pol1-core. Since both Pri1 and Pol1-core are flexibly attached to a stable platform, primer growth produces stress that limits the primer length.
#1: Journal: bioRxiv / Year: 2023
Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase.
Authors: Zuanning Yuan / Roxana Georgescu / Huilin Li / Michael E O'Donnell /
Abstract: The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, ...The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.
History
DepositionDec 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
1: DNA polymerase
A: DNA primase
B: DNA primase large subunit
C: DNA polymerase alpha subunit B
T: template DNA
P: RNA-DNA chimeric primer
hetero molecules


Theoretical massNumber of molelcules
Total (without water)367,2467
Polymers366,8946
Non-polymers3521
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 4 molecules 1ABC

#1: Protein DNA polymerase


Mass: 167027.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4BVQ7
#2: Protein DNA primase


Mass: 47760.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRI1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4C1R0
#3: Protein DNA primase large subunit


Mass: 62348.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRI2, GI527_G0003596 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PVV0
#4: Protein DNA polymerase alpha subunit B


Mass: 78865.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL12, GI527_G0000165 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4F983

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DNA chain / RNA chain / Non-polymers , 3 types, 3 molecules TP

#5: DNA chain template DNA


Mass: 6288.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: RNA chain RNA-DNA chimeric primer


Mass: 4603.852 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


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

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Details

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 alpha/primase complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1160GATAN K3 BIOQUANTUM (6k x 4k)
2150GATAN K3 BIOQUANTUM (6k x 4k)
3150GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
Image processing
IDImage recording-ID
11
22
33
41
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
33PHASE FLIPPING AND AMPLITUDE CORRECTION
44PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 542937 / Symmetry type: POINT
RefinementHighest resolution: 3.56 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0120019
ELECTRON MICROSCOPYf_angle_d1.00527243
ELECTRON MICROSCOPYf_dihedral_angle_d8.0072922
ELECTRON MICROSCOPYf_chiral_restr0.0423018
ELECTRON MICROSCOPYf_plane_restr0.0063414

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