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- EMDB-13512: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) c... -

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Basic information

Entry
Database: EMDB / ID: EMD-13512
TitleCryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation II) - full complex (sharpened map)
Map dataCryo-EM map (sharpened) for the full complex (budding yeast CMG-Csm3-Tof1-Mrc1-Ctf4-PolE-SCF(Dia2)) on double-stranded DNA. Conformation II
Sample
  • Complex: Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation II)
Function / homology
Function and homology information


cellular response to bleomycin / DNA secondary structure 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 / epsilon DNA polymerase complex ...cellular response to bleomycin / DNA secondary structure 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 / epsilon DNA polymerase complex / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / cellular response to cisplatin / cellular response to hydroxyurea / nuclear origin of replication recognition complex / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / anaphase-promoting complex binding / alpha DNA polymerase:primase complex / activation of protein kinase activity / nucleotide-excision repair, DNA gap filling / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / mitotic DNA replication / cullin-RING ubiquitin ligase complex / DNA replication checkpoint signaling / CMG complex / cellular response to chemical stress / regulation of phosphorylation / DNA replication proofreading / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / DNA replication preinitiation complex / single-stranded DNA 3'-5' DNA exonuclease activity / VCB complex / MCM complex / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / elongin complex / replication fork protection complex / mitotic DNA replication checkpoint signaling / positive regulation of protein autoubiquitination / double-strand break repair via break-induced replication / protein neddylation / mitotic DNA replication initiation / mitotic intra-S DNA damage checkpoint signaling / NEDD8 ligase activity / negative regulation of response to oxidative stress / Cul5-RING ubiquitin ligase complex / regulation of DNA-templated DNA replication initiation / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / SCF ubiquitin ligase complex / inner cell mass cell proliferation / Cul2-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / ubiquitin-ubiquitin ligase activity / negative regulation of type I interferon production / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / positive regulation of double-strand break repair / branching morphogenesis of an epithelial tube / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / cochlea development / Apoptotic cleavage of cellular proteins / negative regulation of mitophagy / Prolactin receptor signaling / leading strand elongation / G1/S-Specific Transcription / mitotic G2 DNA damage checkpoint signaling / replication fork processing / nuclear replication fork / cullin family protein binding / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / DNA replication origin binding / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / protein monoubiquitination / PCNA-Dependent Long Patch Base Excision Repair / Activation of the pre-replicative complex / DNA replication initiation / embryonic organ development / Tat-mediated elongation of the HIV-1 transcript / error-prone translesion synthesis / Formation of HIV-1 elongation complex containing HIV-1 Tat / positive regulation of double-strand break repair via homologous recombination / Formation of HIV elongation complex in the absence of HIV Tat / protein K48-linked ubiquitination / Activation of ATR in response to replication stress / response to UV / RNA Polymerase II Transcription Elongation / Nuclear events stimulated by ALK signaling in cancer / Formation of RNA Pol II elongation complex / base-excision repair, gap-filling / RNA Polymerase II Pre-transcription Events / regulation of cellular response to insulin stimulus / positive regulation of TORC1 signaling
Similarity search - Function
DNA polymerase epsilon subunit B, N-terminal / DNA polymerases epsilon N terminal / : / WDHD1 HMG box / WDHD1 first WD40 domain / Claspin / Timeless, C-terminal / Timeless PAB domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 ...DNA polymerase epsilon subunit B, N-terminal / DNA polymerases epsilon N terminal / : / WDHD1 HMG box / WDHD1 first WD40 domain / Claspin / Timeless, C-terminal / Timeless PAB domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Zinc finger domain of DNA polymerase-epsilon / DNA polymerase epsilon, subunit B / Timeless, N-terminal / Timeless / Timeless protein / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / DNA replication licensing factor MCM2-like, winged-helix domain / : / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / : / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / : / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / MCM3-like, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / Cullin protein neddylation domain / MCM N-terminal domain / MCM N-terminal domain / : / Cullin, conserved site / Elongin-C / Cullin family signature. / Elongin B / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Leucine Rich Repeat / Cullin family / Cullin family profile. / HMG boxes A and B DNA-binding domains profile. / DNA polymerase family B, thumb domain / high mobility group / High mobility group box domain / High mobility group box domain superfamily / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain
Similarity search - Domain/homology
Cell division control protein 45 homolog / WD repeat and HMG-box DNA-binding protein 1 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA polymerase epsilon subunit 2 / E3 ubiquitin-protein ligase RBX1 / DNA polymerase epsilon catalytic subunit A ...Cell division control protein 45 homolog / WD repeat and HMG-box DNA-binding protein 1 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA polymerase epsilon subunit 2 / E3 ubiquitin-protein ligase RBX1 / DNA polymerase epsilon catalytic subunit A / Cullin-2 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein PSF1 / Elongin-C / Elongin-B / Leucine-rich repeat protein 1 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF3 / TIMELESS-interacting protein / Claspin / Protein timeless homolog / DNA replication complex GINS protein PSF2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsJenkyn-Bedford M / Yeeles JTP / Deegan TD
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/12 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UU_12016/13 United Kingdom
Wellcome Trust204678/Z/16/Z United Kingdom
CitationJournal: 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
DepositionSep 1, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0152
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0152
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13512.png

Downloads & links

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Map

FileDownload / File: emd_13512.map.gz / Format: CCP4 / Size: 202.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map (sharpened) for the full complex (budding yeast CMG-Csm3-Tof1-Mrc1-Ctf4-PolE-SCF(Dia2)) on double-stranded DNA. Conformation II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 376 pix.
= 398.56 Å		1.06 Å/pix.
x 376 pix.
= 398.56 Å		1.06 Å/pix.
x 376 pix.
= 398.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0152 / Movie #1: 0.0152
Minimum - Maximum-0.031640522 - 0.06619857
Average (Standard dev.)0.00023643812 (±0.0017390195)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions376376376
Spacing376376376
CellA=B=C: 398.55997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z376376376
origin x/y/z0.0000.0000.000
length x/y/z398.560398.560398.560
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ440440440
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS376376376
D min/max/mean-0.0320.0660.000

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Supplemental data

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Sample components

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Entire : Budding yeast replisome on double-stranded DNA engaged with SCF(D...

EntireName: Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation II)
Components
  • Complex: Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation II)

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Supramolecule #1: Budding yeast replisome on double-stranded DNA engaged with SCF(D...

SupramoleculeName: Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation II)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#22
Details: Complex reconstituted in vitro from purified proteins and DNA
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Details: 15 mA
VitrificationCryogen name: ETHANE / Details: Manual plunger.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2160000
CTF correctionSoftware - Name: RELION (ver. 3)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 65390
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3)

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