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- PDB-9mj5: Catalytic domain of human DNA polymerase alpha in complex with DN... -

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Basic information

Entry
Database: PDB / ID: 9mj5
TitleCatalytic domain of human DNA polymerase alpha in complex with DNA and RPA
Components
  • (Replication protein A ...) x 3
  • DNA polymerase alpha catalytic subunit
  • DNA template (35-mer)
  • RNA-DNA primer (11-mer)
KeywordsReplication/DNA/RNA / DNA replication / Replication-DNA-RNA complex
Function / homology
Function and homology information


protein localization to chromosome / DNA replication factor A complex / DNA replication initiation / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / alpha DNA polymerase:primase complex / Polymerase switching / Processive synthesis on the lagging strand / lateral element ...protein localization to chromosome / DNA replication factor A complex / DNA replication initiation / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / alpha DNA polymerase:primase complex / Polymerase switching / Processive synthesis on the lagging strand / lateral element / regulation of DNA damage checkpoint / single-stranded telomeric DNA binding / G-rich strand telomeric DNA binding / lagging strand elongation / Removal of the Flap Intermediate / chromatin-protein adaptor activity / protein localization to site of double-strand break / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / mitotic DNA replication initiation / DNA replication, synthesis of primer / Removal of the Flap Intermediate from the C-strand / HDR through Single Strand Annealing (SSA) / regulation of double-strand break repair via homologous recombination / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / telomeric DNA binding / Impaired BRCA2 binding to RAD51 / leading strand elongation / G1/S-Specific Transcription / hemopoiesis / Presynaptic phase of homologous DNA pairing and strand exchange / site of DNA damage / DNA replication origin binding / PCNA-Dependent Long Patch Base Excision Repair / DNA replication initiation / Regulation of HSF1-mediated heat shock response / Activation of the pre-replicative complex / HSF1 activation / telomere maintenance via telomerase / mismatch repair / Activation of ATR in response to replication stress / SUMOylation of DNA damage response and repair proteins / mitotic G1 DNA damage checkpoint signaling / homeostasis of number of cells within a tissue / telomere maintenance / regulation of mitotic cell cycle / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / Defective pyroptosis / meiotic cell cycle / male germ cell nucleus / nucleotide-excision repair / Fanconi Anemia Pathway / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / double-strand break repair via homologous recombination / base-excision repair / PML body / G2/M DNA damage checkpoint / HDR through Homologous Recombination (HRR) / double-strand break repair via nonhomologous end joining / Dual Incision in GG-NER / Meiotic recombination / DNA-templated DNA replication / Formation of Incision Complex in GG-NER / nuclear matrix / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / nuclear envelope / single-stranded DNA binding / regulation of cell population proliferation / site of double-strand break / Processing of DNA double-strand break ends / protein phosphatase binding / DNA recombination / Regulation of TP53 Activity through Phosphorylation / in utero embryonic development / DNA-directed DNA polymerase / damaged DNA binding / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / nuclear body / DNA repair / nucleotide binding / positive regulation of cell population proliferation / DNA damage response / ubiquitin protein ligase binding / chromatin binding / protein kinase binding / chromatin / nucleolus / enzyme binding / DNA binding / zinc ion binding
Similarity search - Function
Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor-A protein 1, N-terminal domain / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain ...Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor-A protein 1, N-terminal domain / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain / Replication protein A OB domain / : / Replication factor A, C-terminal / Replication factor-A C terminal domain / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / DNA polymerase alpha catalytic subunit / Replication protein A 32 kDa subunit / Replication protein A 70 kDa DNA-binding subunit / Replication protein A 14 kDa subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBaranovskiy, A.G. / Morstadt, L.M. / Romero, E.E. / Babayeva, N.D. / Tahirov, T.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM152032 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: The human primosome requires replication protein A when copying DNA with inverted repeats.
Authors: Andrey G Baranovskiy / Lucia M Morstadt / Eduardo E Romero / Nigar D Babayeva / Tahir H Tahirov /
Abstract: The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric RNA-DNA primers for loading DNA ...The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric RNA-DNA primers for loading DNA polymerases delta and epsilon (Polϵ). Replication protein A (RPA) tightly binds to single-stranded DNA strands, protecting them from nucleolytic digestion and unauthorized transactions. We report here that RPA plays a critical role for the human primosome during DNA synthesis across inverted repeats prone to hairpin formation. On other alternatively structured DNA, forming a G-quadruplex, RPA does not assist primosome. A stimulatory effect of RPA on DNA synthesis across hairpins was also observed for the catalytic domain of Polα but not of Polϵ. The winged helix-turn-helix domain of RPA is essential for an efficient hairpin bypass and increases RPA-Polα cooperativity on the primed DNA template. Cryo-EM studies revealed that this domain is mainly responsible for the interaction between RPA and Polα. The flexible mode of RPA-Polα interaction during DNA synthesis implies the mechanism of template handover between them when the hairpin formation should be avoided. This work provides insight into a cooperative action of RPA and primosome on DNA, which is critical for DNA synthesis across inverted repeats.
History
DepositionDec 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 2.0Mar 12, 2025Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Refinement description / Structure summary
Category: atom_site / em_admin ...atom_site / em_admin / em_software / entity / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_contact_author / pdbx_struct_conn_angle / pdbx_validate_torsion / refine / refine_ls_restr / struct_conn
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.occupancy / _em_admin.last_update / _em_software.version / _entity.pdbx_mutation / _ndb_struct_na_base_pair.buckle / _ndb_struct_na_base_pair.opening / _ndb_struct_na_base_pair.propeller / _ndb_struct_na_base_pair.shear / _ndb_struct_na_base_pair.stagger / _ndb_struct_na_base_pair_step.helical_rise / _ndb_struct_na_base_pair_step.helical_twist / _ndb_struct_na_base_pair_step.inclination / _ndb_struct_na_base_pair_step.rise / _ndb_struct_na_base_pair_step.roll / _ndb_struct_na_base_pair_step.shift / _ndb_struct_na_base_pair_step.slide / _ndb_struct_na_base_pair_step.tilt / _ndb_struct_na_base_pair_step.tip / _ndb_struct_na_base_pair_step.twist / _ndb_struct_na_base_pair_step.x_displacement / _ndb_struct_na_base_pair_step.y_displacement / _pdbx_validate_torsion.phi / _pdbx_validate_torsion.psi / _refine.B_iso_mean / _refine.pdbx_ls_cross_valid_method / _refine.pdbx_stereochemistry_target_values / _refine_ls_restr.dev_ideal / _refine_ls_restr.number / _refine_ls_restr.type
Description: Ligand geometry / Details: we corrected coordinates of zinc ion / Provider: author / Type: Coordinate replacement
Revision 1.1Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / EM metadata
Group: Data processing / Experimental summary / Structure summary
Data content type: EM metadata / EM metadata / EM metadata / Category: em_admin / em_software / entity / Data content type: EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _em_software.version / _entity.pdbx_mutation
Revision 2.1Sep 3, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replication protein A 14 kDa subunit
B: Replication protein A 32 kDa subunit
C: Replication protein A 70 kDa DNA-binding subunit
P: RNA-DNA primer (11-mer)
S: DNA polymerase alpha catalytic subunit
T: DNA template (35-mer)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)179,3319
Polymers178,7746
Non-polymers5573
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Replication protein A ... , 3 types, 3 molecules ABC

#1: Protein Replication protein A 14 kDa subunit / RP-A p14 / Replication factor A protein 3 / RF-A protein 3


Mass: 13583.714 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPA3, REPA3, RPA14 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P35244
#2: Protein Replication protein A 32 kDa subunit / RP-A p32 / Replication factor A protein 2 / RF-A protein 2 / Replication protein A 34 kDa subunit / RP-A p34


Mass: 25931.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPA2, REPA2, RPA32, RPA34 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P15927
#3: Protein Replication protein A 70 kDa DNA-binding subunit / RP-A p70 / Replication factor A protein 1 / RF-A protein 1 / Single-stranded DNA-binding protein


Mass: 21031.748 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPA1, REPA1, RPA70 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P27694

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DNA/RNA hybrid / Protein / DNA chain , 3 types, 3 molecules PST

#4: DNA/RNA hybrid RNA-DNA primer (11-mer)


Mass: 3505.170 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Protein DNA polymerase alpha catalytic subunit / DNA polymerase alpha catalytic subunit p180


Mass: 103957.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA1, POLA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P09884, DNA-directed DNA polymerase
#6: DNA chain DNA template (35-mer)


Mass: 10764.963 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 3 types, 3 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#9: Chemical ChemComp-DCP / 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE


Mass: 467.157 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O13P3

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Details

Has ligand of interestN
Has protein modificationN

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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: Ternary complex of DNA polymerase alpha with DNA and replication protein A
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18529 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312242
ELECTRON MICROSCOPYf_angle_d0.59616742
ELECTRON MICROSCOPYf_dihedral_angle_d16.2782046
ELECTRON MICROSCOPYf_chiral_restr0.0431890
ELECTRON MICROSCOPYf_plane_restr0.0041995

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