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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 | ||||||||||||
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Title | Cryo-EM structure of S. cerevisiae DNA polymerase alpha-primase complex in the DNA elongation state | ||||||||||||
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![]() | DNA BINDING PROTEIN / DNA polymerase / primase / DNA replication | ||||||||||||
Function / homology | ![]() alpha DNA polymerase:primase complex / DNA primase activity / lagging strand elongation / DNA replication, synthesis of primer / 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 / lagging strand elongation / DNA replication, synthesis of primer / 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 / chromatin binding / nucleotide binding / DNA binding / metal ion binding Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() synthetic construct (others) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å | ||||||||||||
![]() | Yuan, Z. / Georgescu, R. / Li, H. / O'Donnell, M. | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 451 KB | Display | ![]() |
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PDB format | ![]() | 344.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.1 MB | Display | |
Data in XML | ![]() | 69.3 KB | Display | |
Data in CIF | ![]() | 104.3 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 29352MC ![]() 8focC ![]() 8fodC ![]() 8foeC ![]() 8fohC ![]() 8fojC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 4 types, 4 molecules 1ABC
#1: Protein | Mass: 167027.766 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: POL1 / Production host: ![]() ![]() |
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#2: Protein | Mass: 47760.367 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PRI1 / Production host: ![]() ![]() |
#3: Protein | Mass: 62348.551 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: PRI2, GI527_G0003596 / Production host: ![]() ![]() |
#4: Protein | Mass: 78865.938 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: POL12, GI527_G0000165 / Production host: ![]() ![]() |
-DNA chain / RNA chain / Non-polymers , 3 types, 3 molecules TP![](data/chem/img/SF4.gif)
![](data/chem/img/SF4.gif)
#5: DNA chain | Mass: 6288.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#6: RNA chain | Mass: 4603.852 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#7: Chemical | ChemComp-SF4 / |
-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: DNA polymerase alpha/primase complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT |
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Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company | ||||||||||||||||
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Microscopy | Model: FEI TITAN KRIOS | ||||||||||||||||
Electron gun | Electron source: ![]() | ||||||||||||||||
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm | ||||||||||||||||
Image recording |
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Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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Image processing |
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CTF correction |
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3D reconstruction | Resolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 542937 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Highest resolution: 3.56 Å | ||||||||||||||||||||||||
Refine LS restraints |
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