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- PDB-5u8s: Structure of eukaryotic CMG helicase at a replication fork -

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Entry
Database: PDB / ID: 5u8s
TitleStructure of eukaryotic CMG helicase at a replication fork
Components
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45
  • DNA (26-MER)
  • DNA (5'-D(P*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*T)-3')
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / CMG helicase / replisome / origin initiation / DNA polymerase / DNA replication
Function / homology
Function and homology information


Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding / GINS complex / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication ...Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding / GINS complex / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / replication fork protection complex / mitotic DNA replication initiation / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / heterochromatin formation / DNA helicase activity / helicase activity / DNA-templated DNA replication / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus ...CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain 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 / MCM4, winged helix domain / DNA replication licensing factor Mcm5 / 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 / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal 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 / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 ...ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein SLD5 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsLi, H. / Li, B. / Georgescu, R. / Yuan, Z. / Santos, R. / Sun, J. / Zhang, D. / Yurieva, O. / O'Donnell, M.E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111472 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115809 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation.
Authors: Roxana Georgescu / Zuanning Yuan / Lin Bai / Ruda de Luna Almeida Santos / Jingchuan Sun / Dan Zhang / Olga Yurieva / Huilin Li / Michael E O'Donnell /
Abstract: The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ...The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.
History
DepositionDec 14, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2017Provider: repository / Type: Initial release
Revision 1.1Feb 1, 2017Group: Database references
Revision 1.2Feb 8, 2017Group: Database references
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Data collection / Category: em_image_scans / pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Nov 8, 2017Group: Derived calculations / Category: pdbx_struct_assembly
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.5Oct 3, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.6Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: DNA replication complex GINS protein PSF1
B: DNA replication complex GINS protein PSF2
C: DNA replication complex GINS protein PSF3
D: DNA replication complex GINS protein SLD5
E: Cell division control protein 45
F: DNA (26-MER)
G: DNA (5'-D(P*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*T)-3')
2: DNA replication licensing factor MCM2
3: DNA replication licensing factor MCM3
4: DNA replication licensing factor MCM4
5: Minichromosome maintenance protein 5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)799,67216
Polymers798,15013
Non-polymers1,5223
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area69120 Å2
ΔGint-304 kcal/mol
Surface area233760 Å2

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Components

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DNA replication complex GINS protein ... , 4 types, 4 molecules ABCD

#1: Protein DNA replication complex GINS protein PSF1 / Partner of Sld five 1


Mass: 24230.576 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12488
#2: Protein DNA replication complex GINS protein PSF2 / Partner of Sld five 2


Mass: 25096.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PSF2, YJL072C, HRF213, J1086 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40359
#3: Protein DNA replication complex GINS protein PSF3 / Partner of Sld five 3


Mass: 21977.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PSF3, YOL146W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12146
#4: Protein DNA replication complex GINS protein SLD5


Mass: 33983.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SLD5, YDR489W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03406

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Protein , 2 types, 2 molecules E5

#5: Protein Cell division control protein 45


Mass: 74324.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08032
#11: Protein Minichromosome maintenance protein 5 / / Cell division control protein 46


Mass: 86505.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29496, DNA helicase

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DNA chain , 2 types, 2 molecules FG

#6: DNA chain DNA (26-MER)


Mass: 7930.117 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: fork DNA_a (26-MER) / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (5'-D(P*AP*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*T)-3')


Mass: 4279.804 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: fork DNA-b (14-mer) / Source: (synth.) synthetic construct (others)

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DNA replication licensing factor ... , 5 types, 5 molecules 23467

#8: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2


Mass: 98911.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29469, DNA helicase
#9: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 107653.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase
#10: Protein DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105096.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30665, DNA helicase
#12: Protein DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase
#13: Protein DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 95049.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38132, DNA helicase

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Non-polymers , 1 types, 3 molecules

#14: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CMG-biotinylated fork DNA-Streptavidin / Type: COMPLEX / Entity ID: #1-#13 / Source: RECOMBINANT
Molecular weightValue: 0.75 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: pRS403,pRS402,pRS404,pRS405,pRS406
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 10 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.9_1692 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 243796 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementResolution: 6.101→332.8 Å / SU ML: 1.98 / Phase error: 57.46 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.3913 2013 0.59 %
Rwork0.4061 --
obs0.406 338881 99.7 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00341768
ELECTRON MICROSCOPYf_angle_d0.92656620
ELECTRON MICROSCOPYf_dihedral_angle_d12.09615855
ELECTRON MICROSCOPYf_chiral_restr0.0326552
ELECTRON MICROSCOPYf_plane_restr0.0037116
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
6.1005-6.25310.06631440.057524210ELECTRON MICROSCOPY100
6.2531-6.42220.55191310.537123911ELECTRON MICROSCOPY100
6.4222-6.61120.54541560.511823791ELECTRON MICROSCOPY100
6.6112-6.82460.58781440.505324243ELECTRON MICROSCOPY100
6.8246-7.06850.56221440.5124304ELECTRON MICROSCOPY100
7.0685-7.35150.50761680.49824158ELECTRON MICROSCOPY100
7.3515-7.68620.48091320.48624117ELECTRON MICROSCOPY100
7.6862-8.09140.44211200.461124044ELECTRON MICROSCOPY100
8.0914-8.59840.45631680.424324036ELECTRON MICROSCOPY100
8.5984-9.26230.43271320.393924317ELECTRON MICROSCOPY100
9.2623-10.19450.38581440.330824141ELECTRON MICROSCOPY100
10.1945-11.66970.31541080.297123753ELECTRON MICROSCOPY99
11.6697-14.70270.29261540.318124003ELECTRON MICROSCOPY99
14.7027-333.29610.27061680.308223840ELECTRON MICROSCOPY99

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