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- PDB-9f27: Solution structure of the Pyrococcus abyssi Rpa2 winged-helix domain -

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Basic information

Entry
Database: PDB / ID: 9f27
TitleSolution structure of the Pyrococcus abyssi Rpa2 winged-helix domain
ComponentsRPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
KeywordsREPLICATION / Winged-helix domain / Polymerase PolD interaction / Primase PriSL interaction / Disordered linker
Function / homologyReplication factor A protein-like / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / DNA binding / RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
Function and homology information
Biological speciesPyrococcus abyssi GE5 (archaea)
MethodSOLUTION NMR / torsion angle dynamics
AuthorsLe Meur, R.A. / Madru, C. / Cordier, F. / Sauguet, L. / Guijarro, J.I.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2024
Title: Communication between DNA polymerases and Replication Protein A within the archaeal replisome.
Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J ...Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J Iñaki Guijarro / Ghislaine Henneke / Ludovic Sauguet /
Abstract: Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. ...Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrate that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveil how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA is shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.
History
DepositionApr 22, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination


Theoretical massNumber of molelcules
Total (without water)11,0061
Polymers11,0061
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: NMR relaxation study, NMR self-diffusion and 15N relaxation indicate a monomer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 200structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 11006.380 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The three N-terminal residues GTG result from cloning and do not belong to RPA
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Gene: PAB2165 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star / References: UniProt: Q9V1Z1
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N HSQC
121isotropic12D 1H-13C HSQC
131isotropic13D HN(CA)CB
141isotropic13D HNCA
151isotropic13D HNCO
181isotropic13D HN(CO)CA
171isotropic13D C(CO)NH
161isotropic13D H(CCO)NH
1101isotropic13D (H)CCH-TOCSY
191isotropic13D HBHA(CO)NH
1111isotropic13D HBHAN
1141isotropic13D 1H-15N NOESY
1131isotropic13D 1H-13C NOESY aliphatic
1151isotropic13D 1H-15N TOCSY
1161isotropic12D CB(CGCD)HD
1171isotropic12D CB(CGCDCE)HE

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Sample preparation

DetailsType: solution
Contents: 300 uM [U-98% 13C; U-98% 15N] RPA2WH, 20 mM MES, 150 mM NaCl, 95% H2O/5% D2O
Label: 1 / Solvent system: 95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
300 uMRPA2WH[U-98% 13C; U-98% 15N]1
20 mMMESnatural abundance1
150 mMNaClnatural abundance1
Sample conditionsIonic strength: 170 mM / Label: 1 / pH: 6 / Pressure: 1 atm / Temperature: 308.15 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE NEO / Manufacturer: Bruker / Model: AVANCE NEO / Field strength: 800 MHz / Details: Equipped with a cryogenically cooled TCI probe

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin4.1.3Bruker Biospincollection
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
CcpNmr Analysis2.5.2CCPNchemical shift assignment
CcpNmr AnalysisCCPNpeak picking
CNS1.2.1Brunger, Adams, Clore, Gros, Nilges and Readstructure calculation
ARIA2.3.2Linge, O'Donoghue and Nilgeschemical shift calculation
CNS1.2.1Brunger, Adams, Clore, Gros, Nilges and Readrefinement
RefinementMethod: torsion angle dynamics / Software ordinal: 3
Details: Structures are calculated using a log-harmonic potential with a total of 1106 restraints: 954 distances, 32 hydrogen bonds and 120 backbone dihedral angles
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 200 / Conformers submitted total number: 10

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