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Yorodumi- EMDB-13496: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) c... -
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-Basic information
Entry | Database: EMDB / ID: EMD-13496 | ||||||||||||
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Title | Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation I) - full complex (sharpened map) | ||||||||||||
Map data | Cryo-EM map (sharpened) for the full complex (budding yeast CMG-Csm3-Tof1-Mrc1-Ctf4-PolE-SCF(Dia2)) on double-stranded DNA. Conformation I | ||||||||||||
Sample |
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Function / homology | Function and homology information cellular response to bleomycin / DNA secondary structure binding / Switching of origins to a post-replicative state / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Unwinding of DNA / cell cycle phase transition / cellular response to cisplatin / DNA replication initiation ...cellular response to bleomycin / DNA secondary structure binding / Switching of origins to a post-replicative state / detection of abiotic stimulus / replication fork arrest / regulation of nuclear cell cycle DNA replication / Unwinding of DNA / cell cycle phase transition / cellular response to cisplatin / DNA replication initiation / epsilon DNA polymerase complex / GINS complex / DNA strand elongation involved in mitotic DNA replication / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / cellular response to hydroxyurea / alpha DNA polymerase:primase complex / mitotic DNA replication / cullin-RING-type E3 NEDD8 transferase / cellular response to chemical stress / anaphase-promoting complex binding / NEDD8 transferase activity / CMG complex / cullin-RING ubiquitin ligase complex / DNA replication checkpoint signaling / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / single-stranded DNA 3'-5' DNA exonuclease activity / MCM complex / DNA replication preinitiation complex / regulation of phosphorylation / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / target-directed miRNA degradation / elongin complex / VCB complex / mitotic DNA replication checkpoint signaling / positive regulation of protein autoubiquitination / replication fork protection complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / protein neddylation / NEDD8 ligase activity / mitotic intra-S DNA damage checkpoint signaling / Cul5-RING ubiquitin ligase complex / negative regulation of response to oxidative stress / regulation of DNA-templated DNA replication initiation / positive regulation of double-strand break repair / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / ubiquitin-ubiquitin ligase activity / Cul4A-RING E3 ubiquitin ligase complex / SCF ubiquitin ligase complex / inner cell mass cell proliferation / Cul2-RING ubiquitin ligase complex / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / ubiquitin ligase complex scaffold activity / Cul4B-RING E3 ubiquitin ligase complex / negative regulation of type I interferon production / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / DNA strand elongation involved in DNA replication / Cul3-RING ubiquitin ligase complex / branching morphogenesis of an epithelial tube / cochlea development / DNA synthesis involved in DNA repair / G1/S-Specific Transcription / activation of protein kinase activity / DNA unwinding involved in DNA replication / replication fork processing / leading strand elongation / Prolactin receptor signaling / Apoptotic cleavage of cellular proteins / nuclear replication fork / protein monoubiquitination / mitotic G2 DNA damage checkpoint signaling / cullin family protein binding / DNA replication origin binding / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / positive regulation of double-strand break repair via homologous recombination / Activation of the pre-replicative complex / DNA replication initiation / PCNA-Dependent Long Patch Base Excision Repair / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / cellular response to interleukin-4 / protein K48-linked ubiquitination / Formation of HIV elongation complex in the absence of HIV Tat / embryonic organ development / error-prone translesion synthesis / Nuclear events stimulated by ALK signaling in cancer / Activation of ATR in response to replication stress / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / response to UV / RNA Polymerase II Pre-transcription Events / positive regulation of TORC1 signaling / T cell activation / DNA helicase activity Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||||||||
Authors | Jenkyn-Bedford M / Yeeles JTP / Deegan TD | ||||||||||||
Funding support | United Kingdom, 3 items
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Citation | Journal: Nature / Year: 2021 Title: A conserved mechanism for regulating replisome disassembly in eukaryotes. Authors: Michael Jenkyn-Bedford / Morgan L Jones / Yasemin Baris / Karim P M Labib / Giuseppe Cannone / Joseph T P Yeeles / Tom D Deegan / Abstract: Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily ...Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCF in budding yeast, CUL2 in metazoa), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA. However, it is unknown how SCF and CUL2 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13496.map.gz | 10.2 MB | EMDB map data format | |
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Header (meta data) | emd-13496-v30.xml emd-13496.xml | 16.6 KB 16.6 KB | Display Display | EMDB header |
Images | emd_13496.png | 107.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13496 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13496 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_13496.map.gz / Format: CCP4 / Size: 202.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM map (sharpened) for the full complex (budding yeast CMG-Csm3-Tof1-Mrc1-Ctf4-PolE-SCF(Dia2)) on double-stranded DNA. Conformation I | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Budding yeast replisome on double-stranded DNA engaged with SCF(D...
Entire | Name: Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation I) |
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Components |
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-Supramolecule #1: Budding yeast replisome on double-stranded DNA engaged with SCF(D...
Supramolecule | Name: Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation I) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#22 Details: Complex reconstituted in vitro from purified proteins and DNA |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Recombinant expression | Organism: Saccharomyces cerevisiae (brewer's yeast) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.6 |
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Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Details: 15 mA |
Vitrification | Cryogen name: ETHANE / Details: Manual plunger. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 12730 / Average exposure time: 4.0 sec. / Average electron dose: 38.8 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |