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- EMDB-49101: Catalytic domain of human DNA polymerase alpha in complex with DN... -

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Basic information

Entry
Database: EMDB / ID: EMD-49101
TitleCatalytic domain of human DNA polymerase alpha in complex with DNA and RPA (consensus map)
Map dataRPAcore/Pola/DNA consensus map
Sample
  • Complex: Ternary complex of DNA polymerase alpha with DNA and replication protein A
KeywordsDNA replication / RPAcore-Pola-DNA-RNA complex / REPLICATION
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: bioRxiv / Year: 2025
Title: Human primosome requires replication protein A when copying DNA with inverted repeats.
Authors: Andrey Baranovskiy / Lucia Morstadt / Eduardo E Romero / Nigar 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
DepositionFeb 6, 2025-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49101.png

Downloads & links

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Map

FileDownload / File: emd_49101.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRPAcore/Pola/DNA consensus map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (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: 0.05
Minimum - Maximum-0.22267886 - 0.4192333
Average (Standard dev.)0.00027333913 (±0.010436742)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Half map: half A map

Fileemd_49101_half_map_1.map
Annotationhalf_A map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half B map

Fileemd_49101_half_map_2.map
Annotationhalf_B map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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

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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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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

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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