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- PDB-8ok2: Bipartite interaction of TOPBP1 with the GINS complex -

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Basic information

Entry
Database: PDB / ID: 8ok2
TitleBipartite interaction of TOPBP1 with the GINS complex
Components
  • DNA replication complex GINS protein PSF1
  • DNA replication complex GINS protein PSF2
  • DNA replication complex GINS protein PSF3
  • DNA replication complex GINS protein SLD5
  • Topoisomerase (DNA) II binding protein 1
KeywordsREPLICATION / Scaffold / Replisome / Recruitment
Function / homology
Function and homology information


Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / CMG complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / inner cell mass cell proliferation / isomerase activity / DNA-templated DNA replication / multicellular organism growth ...Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / CMG complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / inner cell mass cell proliferation / isomerase activity / DNA-templated DNA replication / multicellular organism growth / spindle pole / cellular senescence / chromosome / DNA replication / DNA repair / centrosome / DNA binding / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
: / TopBP1, first BRCT domain / : / TopBP1/SLF1, BRCT domain / Secretoglobin superfamily / : / PSF2 N-terminal domain / : / : / PSF3 N-terminal domain ...: / TopBP1, first BRCT domain / : / TopBP1/SLF1, BRCT domain / Secretoglobin superfamily / : / PSF2 N-terminal domain / : / : / PSF3 N-terminal domain / PSF1 C-terminal domain / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / 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 / GINS complex, subunit Psf1 / GINS, helical bundle-like domain superfamily / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily
Similarity search - Domain/homology
DNA topoisomerase 2-binding protein 1 / DNA replication complex GINS protein PSF1 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsDay, M. / Oliver, A.W. / Pearl, L.H.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Cancer Research UK United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: TopBP1 utilises a bipartite GINS binding mode to support genome replication.
Authors: Matthew Day / Bilal Tetik / Milena Parlak / Yasser Almeida-Hernández / Markus Räschle / Farnusch Kaschani / Heike Siegert / Anika Marko / Elsa Sanchez-Garcia / Markus Kaiser / Isabel A ...Authors: Matthew Day / Bilal Tetik / Milena Parlak / Yasser Almeida-Hernández / Markus Räschle / Farnusch Kaschani / Heike Siegert / Anika Marko / Elsa Sanchez-Garcia / Markus Kaiser / Isabel A Barker / Laurence H Pearl / Antony W Oliver / Dominik Boos /
Abstract: Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies ...Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.
History
DepositionMar 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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: Topoisomerase (DNA) II binding protein 1


Theoretical massNumber of molelcules
Total (without water)147,0185
Polymers147,0185
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, cross-linking, gel filtration, immunoprecipitation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DNA replication complex GINS protein PSF1 / GINS complex subunit 1


Mass: 17772.438 Da / Num. of mol.: 1 / Mutation: V97I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS1, KIAA0186, PSF1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q14691
#2: Protein DNA replication complex GINS protein PSF2 / GINS complex subunit 2


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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y248
#3: Protein DNA replication complex GINS protein PSF3 / GINS complex subunit 3


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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BRX5
#4: Protein DNA replication complex GINS protein SLD5 / GINS complex subunit 4


Mass: 30831.814 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS4, SLD5 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BRT9
#5: Protein Topoisomerase (DNA) II binding protein 1


Mass: 52397.535 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TOPBP1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A7E2X7

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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: GINS complex bound to TOPBP1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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 FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 39.69290657 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154278 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 78.33 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00217578
ELECTRON MICROSCOPYf_angle_d0.585610230
ELECTRON MICROSCOPYf_chiral_restr0.03911123
ELECTRON MICROSCOPYf_plane_restr0.00541332
ELECTRON MICROSCOPYf_dihedral_angle_d13.45742901

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