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Yorodumi- PDB-8b9a: S. cerevisiae replisome + Ctf4, bound by pol alpha primase. Compl... -
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-Basic information
Entry | Database: PDB / ID: 8b9a | ||||||
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Title | S. cerevisiae replisome + Ctf4, bound by pol alpha primase. Complex engaged with a fork DNA substrate containing a 60 nucleotide lagging strand. | ||||||
Components |
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Keywords | REPLICATION / helicase / polymerase / pol alpha / priming | ||||||
Function / homology | Function and homology information Inhibition of replication initiation of damaged DNA by RB1/E2F1 / establishment of sister chromatid cohesion / H3-H4 histone complex chaperone activity / maintenance of DNA repeat elements / Unwinding of DNA / replication fork arrest / Cul8-RING ubiquitin ligase complex / regulation of nuclear cell cycle DNA replication / DNA replication initiation / meiotic chromosome segregation ...Inhibition of replication initiation of damaged DNA by RB1/E2F1 / establishment of sister chromatid cohesion / H3-H4 histone complex chaperone activity / maintenance of DNA repeat elements / Unwinding of DNA / replication fork arrest / Cul8-RING ubiquitin ligase complex / regulation of nuclear cell cycle DNA replication / DNA replication initiation / meiotic chromosome segregation / RNA-templated DNA biosynthetic process / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / DNA strand elongation involved in mitotic DNA replication / MCM complex binding / Processive synthesis on the lagging strand / GINS complex / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / Removal of the Flap Intermediate / premeiotic DNA replication / Polymerase switching / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / telomere capping / anaphase-promoting complex binding / mitotic DNA replication / alpha DNA polymerase:primase complex / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / DNA replication checkpoint signaling / DNA primase activity / establishment of mitotic sister chromatid cohesion / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / primosome complex / DNA replication, synthesis of primer / mitotic DNA replication checkpoint signaling / lagging strand elongation / double-strand break repair via break-induced replication / cellular response to osmotic stress / mitotic DNA replication initiation / single-stranded DNA helicase activity / regulation of DNA-templated DNA replication initiation / mitotic intra-S DNA damage checkpoint signaling / DNA strand elongation involved in DNA replication / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / nuclear chromosome / DNA synthesis involved in DNA repair / DNA biosynthetic process / leading strand elongation / DNA unwinding involved in DNA replication / replication fork processing / nuclear replication fork / mitotic G2 DNA damage checkpoint signaling / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / Ub-specific processing proteases / DNA helicase activity / telomere maintenance / nuclear periphery / replication fork / meiotic cell cycle / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / DNA-templated DNA replication / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / double-strand break repair / nucleosome assembly / nuclear envelope / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA helicase / DNA replication / DNA-directed DNA polymerase / chromosome, telomeric region / DNA-directed DNA polymerase activity / protein stabilization / DNA repair / nucleotide binding / DNA damage response / chromatin binding / ATP hydrolysis activity / mitochondrion / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | ||||||
Authors | Jones, M.L. / Yeeles, J.T.P. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Mol Cell / Year: 2023 Title: 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. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8b9a.cif.gz | 3 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8b9a.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8b9a.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8b9a_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
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Full document | 8b9a_full_validation.pdf.gz | 1.8 MB | Display | |
Data in XML | 8b9a_validation.xml.gz | 194 KB | Display | |
Data in CIF | 8b9a_validation.cif.gz | 314.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/b9/8b9a ftp://data.pdbj.org/pub/pdb/validation_reports/b9/8b9a | HTTPS FTP |
-Related structure data
Related structure data | 15309MC 8b9bC 8b9cC 8b9dC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA replication licensing factor ... , 5 types, 5 molecules 23467
#1: Protein | Mass: 98911.539 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29469, DNA helicase |
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#2: Protein | Mass: 111987.562 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase |
#3: Protein | Mass: 105138.375 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30665, DNA helicase |
#5: Protein | Mass: 113110.211 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase |
#6: Protein | Mass: 95049.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38132, DNA helicase |
-Protein , 8 types, 10 molecules 5AGPSXYHLK
#4: Protein | Mass: 86505.734 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29496, DNA helicase |
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#7: Protein | 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, YKL045W, YKL258 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P20457 |
#13: Protein | Mass: 75154.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08032 |
#15: Protein | Mass: 126078.859 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: MRC1, YCL061C, YCL61C/YCL60C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25588 |
#18: Protein | Mass: 51827.090 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PRI1, YIR008C, YIB8C / Production host: Saccharomyces cerevisiae (brewer's yeast) References: UniProt: P10363, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases |
#19: Protein | Mass: 141296.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: TOF1, YNL273W, N0636 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53840 |
#20: Protein | Mass: 36588.758 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: CSM3, YMR048W, YM9796.01 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04659 |
#21: Protein | Mass: 108610.148 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: CTF4, CHL15, POB1, YPR135W, P9659.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q01454 |
-DNA polymerase alpha ... , 2 types, 2 molecules BJ
#8: Protein | 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, YBL035C, YBL0414 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38121 |
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#14: Protein | 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, CDC17, YNL102W, N2181 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P13382, DNA-directed DNA polymerase |
-DNA replication complex GINS protein ... , 4 types, 4 molecules CDEF
#9: Protein | Mass: 24230.576 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12488 |
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#10: Protein | Mass: 25096.807 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PSF2, YJL072C, HRF213, J1086 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40359 |
#11: Protein | Mass: 24437.859 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PSF3, YOL146W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12146 |
#12: Protein | Mass: 33983.617 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: SLD5, YDR489W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03406 |
-DNA chain , 2 types, 2 molecules QR
#16: DNA chain | Mass: 26092.645 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) |
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#17: DNA chain | Mass: 32046.451 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) |
-Non-polymers , 3 types, 12 molecules
#22: Chemical | ChemComp-ANP / #23: Chemical | ChemComp-MG / #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 |
-Sample preparation
Component | Name: S. cerevisiae replisome + Ctf4, bound by pol alpha primase. Complex engaged with a fork DNA substrate containing a 60 nucleotide lagging strand. Type: COMPLEX / Entity ID: #1-#13, #18, #21, #15-#17, #19-#20, #14 / Source: RECOMBINANT |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Source (recombinant) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Buffer solution | pH: 7.6 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-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: 39.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-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: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54970 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 97.22 Å2 | ||||||||||||||||||||||||
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