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Open data
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Basic information
| Entry | Database: PDB / ID: 8b9d | ||||||
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| Title | Human replisome bound by Pol Alpha | ||||||
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Keywords | REPLICATION / human / priming / polymerase | ||||||
| Function / homology | Function and homology informationcellular response to bleomycin / DNA primase AEP / DNA secondary structure binding / ribonucleotide binding / detection of abiotic stimulus / regulation of nuclear cell cycle DNA replication / replication fork arrest / Switching of origins to a post-replicative state / cell cycle phase transition / Unwinding of DNA ...cellular response to bleomycin / DNA primase AEP / DNA secondary structure binding / ribonucleotide binding / detection of abiotic stimulus / regulation of nuclear cell cycle DNA replication / replication fork arrest / Switching of origins to a post-replicative state / cell cycle phase transition / Unwinding of DNA / DNA replication initiation / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / activation of protein kinase activity / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / cellular response to cisplatin / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / replication fork protection complex / nuclear origin of replication recognition complex / cellular response to hydroxyurea / regulation of type I interferon production / alpha DNA polymerase:primase complex / anaphase-promoting complex binding / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / mitotic DNA replication / DNA replication checkpoint signaling / Polymerase switching / regulation of phosphorylation / CMG complex / Processive synthesis on the lagging strand / DNA replication preinitiation complex / MCM complex / lagging strand elongation / Removal of the Flap Intermediate / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / Polymerase switching on the C-strand of the telomere / mitotic DNA replication initiation / DNA replication, synthesis of primer / mitotic intra-S DNA damage checkpoint signaling / regulation of DNA-templated DNA replication initiation / inner cell mass cell proliferation / DNA strand elongation involved in DNA replication / Apoptotic cleavage of cellular proteins / branching morphogenesis of an epithelial tube / DNA synthesis involved in DNA repair / positive regulation of double-strand break repair / leading strand elongation / G1/S-Specific Transcription / mitotic G2 DNA damage checkpoint signaling / nuclear replication fork / replication fork processing / DNA replication origin binding / cochlea development / DNA replication initiation / Activation of the pre-replicative complex / Activation of ATR in response to replication stress / response to UV / positive regulation of double-strand break repair via homologous recombination / cellular response to interleukin-4 / DNA helicase activity / lung development / cellular response to epidermal growth factor stimulus / DNA damage checkpoint signaling / morphogenesis of an epithelium / Defective pyroptosis / Assembly of the pre-replicative complex / enzyme activator activity / helicase activity / circadian rhythm / regulation of circadian rhythm / peptidyl-serine phosphorylation / double-strand break repair via nonhomologous end joining / DNA-templated DNA replication / multicellular organism growth / cellular response to xenobiotic stimulus / nuclear matrix / Orc1 removal from chromatin / protein import into nucleus / DNA-directed RNA polymerase activity / cellular senescence / nuclear envelope / mitotic cell cycle / nucleosome assembly / single-stranded DNA binding / site of double-strand break / 4 iron, 4 sulfur cluster binding / Processing of DNA double-strand break ends / histone binding / DNA helicase / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / cell population proliferation / Ub-specific processing proteases / cilium / ciliary basal body Similarity search - Function | ||||||
| Biological species | Homo sapiens (human) | ||||||
| Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||
Authors | Jones, M.L. / Yeeles, J.T.P. | ||||||
| Funding support | United Kingdom, 1items
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Citation | Journal: Mol Cell / Year: 2023Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication. Authors: Morgan L Jones / Valentina Aria / Yasemin Baris / Joseph T P Yeeles / ![]() Abstract: During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand ...During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis. | ||||||
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Structure visualization
| Structure viewer | Molecule: Molmil Jmol/JSmol |
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Downloads & links
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Download
| PDBx/mmCIF format | 8b9d.cif.gz | 2.8 MB | Display | PDBx/mmCIF format |
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| PDB format | pdb8b9d.ent.gz | Display | PDB format | |
| PDBx/mmJSON format | 8b9d.json.gz | Tree view | PDBx/mmJSON format | |
| Others | Other downloads |
-Validation report
| Summary document | 8b9d_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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| Full document | 8b9d_full_validation.pdf.gz | 1.6 MB | Display | |
| Data in XML | 8b9d_validation.xml.gz | 184.5 KB | Display | |
| Data in CIF | 8b9d_validation.cif.gz | 304.7 KB | Display | |
| Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/b9/8b9d ftp://data.pdbj.org/pub/pdb/validation_reports/b9/8b9d | HTTPS FTP |
-Related structure data
| Related structure data | ![]() 15341MC ![]() 8b9aC ![]() 8b9bC ![]() 8b9cC M: map data used to model this data C: citing same article ( |
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| Similar structure data | Similarity search - Function & homology F&H Search |
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Assembly
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Components
-DNA replication licensing factor ... , 6 types, 6 molecules 234567
| #1: Protein | Mass: 102034.102 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM2, BM28, CCNL1, CDCL1, KIAA0030 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49736, DNA helicase |
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| #2: Protein | Mass: 91110.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P25205, DNA helicase |
| #3: Protein | Mass: 96684.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM4, CDC21 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33991, DNA helicase |
| #4: Protein | Mass: 82406.633 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM5, CDC46 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33992, DNA helicase |
| #5: Protein | Mass: 93010.273 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM6 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14566, DNA helicase |
| #6: Protein | Mass: 81411.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MCM7, CDC47, MCM2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33993, DNA helicase |
-DNA polymerase alpha ... , 2 types, 2 molecules AB
| #7: Protein | Mass: 66015.539 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLA2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14181 |
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| #21: Protein | Mass: 170392.656 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLA1, POLA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P09884, DNA-directed DNA polymerase |
-Protein , 7 types, 9 molecules CKLPQOHJI
| #8: Protein | Mass: 66016.891 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CDC45, CDC45L, CDC45L2, UNQ374/PRO710 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75419 |
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| #13: Protein | Mass: 138903.031 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TIMELESS, TIM, TIM1, TIMELESS1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9UNS1 |
| #14: Protein | Mass: 34600.223 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TIPIN / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BVW5 |
| #17: Protein | Mass: 58890.918 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49643 |
| #18: Protein | Mass: 155184.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CLSPN / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9HAW4 |
| #19: Protein | Mass: 54242.598 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49642, DNA primase AEP |
| #20: Protein | Mass: 129966.961 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: WDHD1, AND1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75717 |
-DNA replication complex GINS protein ... , 4 types, 4 molecules DEFG
| #9: Protein | Mass: 23022.469 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS1, KIAA0186, PSF1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14691 |
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| #10: Protein | Mass: 21453.713 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS2, PSF2, CGI-122, DC5, HSPC037 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Y248 |
| #11: Protein | Mass: 24562.611 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS3, PSF3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BRX5 |
| #12: Protein | Mass: 29983.041 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GINS4, SLD5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BRT9 |
-DNA chain , 2 types, 2 molecules MN
| #15: DNA chain | Mass: 26092.645 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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| #16: DNA chain | Mass: 26402.895 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
-Non-polymers , 3 types, 10 molecules 




| #22: Chemical | | #23: Chemical | #24: Chemical | ChemComp-ZN / |
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-Details
| Has ligand of interest | Y |
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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: Human replisome bound by pol alpha, engaged on a fork DNA substrate with a 60 nucleotide lagging strand. Type: COMPLEX Entity ID: #19, #7, #21, #17, #1-#6, #8-#12, #20, #13-#16, #18 Source: RECOMBINANT |
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| Molecular weight | Experimental value: NO |
| Source (natural) | Organism: Homo sapiens (human) |
| Source (recombinant) | Organism: Trichoplusia ni (cabbage looper) |
| Buffer solution | pH: 7.6 |
| 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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
| Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm |
| Image recording | Electron dose: 37.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
| EM software | Name: PHENIX / Version: 1.20_4459: / Category: model refinement | ||||||||||||||||||||||||
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| CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
| 3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 174696 / Symmetry type: POINT | ||||||||||||||||||||||||
| Refine LS restraints |
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Homo sapiens (human)
United Kingdom, 1items
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Trichoplusia ni (cabbage looper)
FIELD EMISSION GUN