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- EMDB-48312: Catalytic domain of human DNA polymerase alpha in complex with DN... -

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Entry
Database: EMDB / ID: EMD-48312
TitleCatalytic domain of human DNA polymerase alpha in complex with DNA and RPA
Map datacomposite map, 3.5 angstrom
Sample
  • Complex: Ternary complex of DNA polymerase alpha with DNA and replication protein A
    • Protein or peptide: Replication protein A 14 kDa subunit
    • Protein or peptide: Replication protein A 32 kDa subunit
    • Protein or peptide: Replication protein A 70 kDa DNA-binding subunit
    • Other: RNA-DNA primer (11-mer)
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • DNA: DNA template (35-mer)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE
KeywordsDNA 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBaranovskiy AG / Morstadt LM / Romero EE / Babayeva ND / Tahirov TH
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateSep 3, 2025-
Current statusSep 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48312.png

Downloads & links

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Map

FileDownload / File: emd_48312.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map, 3.5 angstrom
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.72 Å/pix.
x 450 pix.
= 324. Å		0.72 Å/pix.
x 450 pix.
= 324. Å		0.72 Å/pix.
x 450 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-21.874872 - 39.672035000000001
Average (Standard dev.)0.0021560192 (±1.0123504)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Ternary complex of DNA polymerase alpha with DNA and replication ...

EntireName: Ternary complex of DNA polymerase alpha with DNA and replication protein A
Components
  • Complex: Ternary complex of DNA polymerase alpha with DNA and replication protein A
    • Protein or peptide: Replication protein A 14 kDa subunit
    • Protein or peptide: Replication protein A 32 kDa subunit
    • Protein or peptide: Replication protein A 70 kDa DNA-binding subunit
    • Other: RNA-DNA primer (11-mer)
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • DNA: DNA template (35-mer)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: Ternary complex of DNA polymerase alpha with DNA and replication ...

SupramoleculeName: Ternary complex of DNA polymerase alpha with DNA and replication protein A
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Replication protein A 14 kDa subunit

MacromoleculeName: Replication protein A 14 kDa subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.583714 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MVDMMDLPRS RINAGMLAQF IDKPVCFVGR LEKIHPTGKM FILSDGEGKN GTIELMEPLD EEISGIVEVV GRVTAKATIL CTSYVQFKE DSHPFDLGLY NEAVKIIHDF PQFYPLGIVQ HD

UniProtKB: Replication protein A 14 kDa subunit

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Macromolecule #2: Replication protein A 32 kDa subunit

MacromoleculeName: Replication protein A 32 kDa subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.931359 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: AEKKSRARAQ HIVPCTISQL LSATLVDEVF RIGNVEISQV TIVGIIRHAE KAPTNIVYKI DDMTAAPMDV RQWVDTDDTS SENTVVPPE TYVKVAGHLR SFQNKKSLVA FKIMPLEDMN EFTTHILEVI NAHMVLSKAN SQPSAGRAPI SNPGMSEAGN F GGNSFMPA ...String:
AEKKSRARAQ HIVPCTISQL LSATLVDEVF RIGNVEISQV TIVGIIRHAE KAPTNIVYKI DDMTAAPMDV RQWVDTDDTS SENTVVPPE TYVKVAGHLR SFQNKKSLVA FKIMPLEDMN EFTTHILEVI NAHMVLSKAN SQPSAGRAPI SNPGMSEAGN F GGNSFMPA NGLTVAQNQV LNLIKACPRP EGLNFQDLKN QLKHMSVSSI KQAVDFLSNE GHIYSTVDDD HFKSTDAE

UniProtKB: Replication protein A 32 kDa subunit

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Macromolecule #3: Replication protein A 70 kDa DNA-binding subunit

MacromoleculeName: Replication protein A 70 kDa DNA-binding subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.031748 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SNTNWKTLYE VKSENLGQGD KPDYFSSVAT VVYLRKENCM YQACPTQDCN KKVIDQQNGL YRCEKCDTEF PNFKYRMILS VNIADFQEN QWVTCFQESA EAILGQNAAY LGELKDKNEQ AFEEVFQNAN FRSFIFRVRV KVETYNDESR IKATVMDVKP V DYREYGRR LVMSIRRSAL M

UniProtKB: Replication protein A 70 kDa DNA-binding subunit

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Macromolecule #5: DNA polymerase alpha catalytic subunit

MacromoleculeName: DNA polymerase alpha catalytic subunit / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 103.957078 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: ADEEQVFHFY WLDAYEDQYN QPGVVFLFGK VWIESAETHV SCCVMVKNIE RTLYFLPREM KIDLNTGKET GTPISMKDVY EEFDEKIAT KYKIMKFKSK PVEKNYAFEI PDVPEKSEYL EVKYSAEMPQ LPQDLKGETF SHVFGTNTSS LELFLMNRKI K GPCWLEVK ...String:
ADEEQVFHFY WLDAYEDQYN QPGVVFLFGK VWIESAETHV SCCVMVKNIE RTLYFLPREM KIDLNTGKET GTPISMKDVY EEFDEKIAT KYKIMKFKSK PVEKNYAFEI PDVPEKSEYL EVKYSAEMPQ LPQDLKGETF SHVFGTNTSS LELFLMNRKI K GPCWLEVK SPQLLNQPVS WCKVEAMALK PDLVNVIKDV SPPPLVVMAF SMKTMQNAKN HQNEIIAMAA LVHHSFALDK AA PKPPFQS HFCVVSKPKD CIFPYAFKEV IEKKNVKVEV AATERTLLGF FLAKVHKIDP DIIVGHNIYG FELEVLLQRI NVC KAPHWS KIGRLKRSNM PKLGGRSGFG ERNATCGRMI CDVEISAKEL IRCKSYHLSE LVQQILKTER VVIPMENIQN MYSE SSQLL YLLEHTWKDA KFILQIMCEL NVLPLALQIT NIAGNIMSRT LMGGRSERNE FLLLHAFYEN NYIVPDKQIF RKPQQ KLGD EDEEIDGDTN KYKKGRKKAA YAGGLVLDPK VGFYDKFILL LDFNSLYPSI IQEFNICFTT VQRVASEAQK VTEDGE QEQ IPELPDPSLE MGILPREIRK LVERRKQVKQ LMKQQDLNPD LILQYDIRQK ALKLTANSMY GCLGFSYSRF YAKPLAA LV TYKGREILMH TKEMVQKMNL EVIYGDTDSI MINTNSTNLE EVFKLGNKVK SEVNKLYKLL EIDIDGVFKS LLLLKKKK Y AALVVEPTSD GNYVTKQELK GLDIVRRDWC DLAKDTGNFV IGQILSDQSR DTIVENIQKR LIEIGENVLN GSVPVSQFE INKALTKDPQ DYPDKKSLPH VHVALWINSQ GGRKVKAGDT VSYVICQDGS NLTASQRAYA PEQLQKQDNL TIDTQYYLAQ QIHPVVARI CEPIDGIDAV LIATWLGLDP T

UniProtKB: DNA polymerase alpha catalytic subunit

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Macromolecule #4: RNA-DNA primer (11-mer)

MacromoleculeName: RNA-DNA primer (11-mer) / type: other / ID: 4 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.50517 KDa
SequenceString:
GCCUGGAGC(DG) (DC)

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Macromolecule #6: DNA template (35-mer)

MacromoleculeName: DNA template (35-mer) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.764963 KDa
SequenceString:
(DA)(DA)(DT)(DC)(DT)(DA)(DG)(DT)(DA)(DA) (DC)(DA)(DT)(DA)(DG)(DT)(DA)(DT)(DA)(DC) (DA)(DT)(DA)(DG)(DG)(DC)(DG)(DC)(DT) (DC)(DC)(DA)(DG)(DG)(DC)

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #9: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 9 / Number of copies: 1 / Formula: DCP
Molecular weightTheoretical: 467.157 Da
Chemical component information

ChemComp-DCP:
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

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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: 7.7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 18529
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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