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- EMDB-29891: Tetramer core subcomplex (conformation 3) of Xenopus laevis DNA p... -

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Entry
Database: EMDB / ID: EMD-29891
TitleTetramer core subcomplex (conformation 3) of Xenopus laevis DNA polymerase alpha-primase
Map dataTetramer core subcomplex (conformation 3) of Xenopus laevis DNA polymerase alpha-primase
Sample
  • Complex: Polymerase alpha-primase
    • Complex: Polymerase alpha
      • Protein or peptide: POLA1
      • Protein or peptide: POLA2
    • Complex: Primase
      • Protein or peptide: PRIM2
      • Protein or peptide: PRIM1
KeywordsPrimase / DNA polymerase / chimeric RNA-DNA primer / RNA/DNA hybrid / DNA replication / DNA synthesis / REPLICATION
Function / homology
Function and homology information


alpha DNA polymerase:primase complex / DNA primase activity / primosome complex / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication initiation / DNA-directed RNA polymerase complex ...alpha DNA polymerase:primase complex / DNA primase activity / primosome complex / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication initiation / DNA-directed RNA polymerase complex / double-strand break repair via nonhomologous end joining / nuclear matrix / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / nuclear envelope / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / chromatin / nucleolus / DNA binding / nucleoplasm / nucleus / metal ion binding
Similarity search - Function
DNA polymerase alpha, subunit B, N-terminal domain superfamily / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit ...DNA polymerase alpha, subunit B, N-terminal domain superfamily / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / 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 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / 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 / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA primase large subunit / DNA polymerase alpha subunit B / DNA primase / DNA polymerase alpha catalytic subunit
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsMullins EA / Chazin WJ / Eichman BF
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136401 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118089 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA92584 United States
CitationJournal: bioRxiv / Year: 2023
Title: A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.
Authors: Elwood A Mullins / Lauren E Salay / Clarissa L Durie / Noah P Bradley / Jane E Jackman / Melanie D Ohi / Walter J Chazin / Brandt F Eichman
Abstract: The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is ...The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5'-end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.
History
DepositionFeb 22, 2023-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29891.png

Downloads & links

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Map

FileDownload / File: emd_29891.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTetramer core subcomplex (conformation 3) of Xenopus laevis DNA polymerase alpha-primase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.64 Å/pix.
x 200 pix.
= 328. Å		1.64 Å/pix.
x 200 pix.
= 328. Å		1.64 Å/pix.
x 200 pix.
= 328. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.8609122 - 3.4300573
Average (Standard dev.)-0.0033924987 (±0.06910149)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Tetramer core subcomplex (conformation 3) of Xenopus laevis...

Fileemd_29891_half_map_1.map
AnnotationTetramer core subcomplex (conformation 3) of Xenopus laevis DNA polymerase alpha-primase (half 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Tetramer core subcomplex (conformation 3) of Xenopus laevis...

Fileemd_29891_half_map_2.map
AnnotationTetramer core subcomplex (conformation 3) of Xenopus laevis DNA polymerase alpha-primase (half 2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Polymerase alpha-primase

EntireName: Polymerase alpha-primase
Components
  • Complex: Polymerase alpha-primase
    • Complex: Polymerase alpha
      • Protein or peptide: POLA1
      • Protein or peptide: POLA2
    • Complex: Primase
      • Protein or peptide: PRIM2
      • Protein or peptide: PRIM1

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Supramolecule #1: Polymerase alpha-primase

SupramoleculeName: Polymerase alpha-primase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Heterotetrameric protein complex
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: Polymerase alpha

SupramoleculeName: Polymerase alpha / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 / Details: Heterodimer consisting of POLA1 and POLA2
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 200 KDa

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Supramolecule #3: Primase

SupramoleculeName: Primase / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4 / Details: Heterodimer consisting of PRIM1 and PRIM2
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: POLA1

MacromoleculeName: POLA1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
SequenceString: SNAADGSQVF RFYWLDAYED QYSQPGVVYL FGKVWIESAD AYVSCCVSVK NIERTVYLLP RENRVQLSTG KDTGAPVSMM HVYQEFNEAV AEKYKIMKFK SKKVDKDYAF EIPDVPASSE YLEVRYSADS PQLPQDLKGE TFSHVFGTNT SSLELFLLSR KIKGPSWLEI ...String:
SNAADGSQVF RFYWLDAYED QYSQPGVVYL FGKVWIESAD AYVSCCVSVK NIERTVYLLP RENRVQLSTG KDTGAPVSMM HVYQEFNEAV AEKYKIMKFK SKKVDKDYAF EIPDVPASSE YLEVRYSADS PQLPQDLKGE TFSHVFGTNT SSLELFLLSR KIKGPSWLEI KSPQLSSQPM SWCKVEAVVT RPDQVSVVKD LAPPPVVVLS LSMKTVQNAK THQNEIVAIA ALVHHTFPLD KAPPQPPFQT HFCVLSKLND CIFPYDYNEA VKQKNANIEI ALTERTLLGF FLAKIHKIDP DVIVGHDIYG FDLEVLLQRI NSCKVPFWSK IGRLRRSVMP KLGGRSGFAE RNAACGRIIC DIEISAKELI RCKSYHLSEL VHQILKAERV VIPPENIRNA YNDSVHLLYM LENTWIDAKF ILQIMCELNV LPLALQITNI AGNVMSRTLM GGRSERNEYL LLHAFTENNF IVPDKPVFKK MQQTTVEDND DMGTDQNKNK SRKKAAYAGG LVLEPKVGFY DKFILLLDFN SLYPSIIQEY NICFTTVHRE APSTQKGEDQ DEIPELPHSD LEMGILPREI RKLVERRRHV KQLMKQPDLN PDLYLQYDIR QKALKLTANS MYGCLGFSYS RFYAKPLAAL VTHQGREILL HTKEMVQKMN LEVIYGDTDS IMINTNCNNL EEVFKLGNRV KSEINKSYKL LEIDIDGIFK SLLLLKKKKY AALTVEPTGD GKYVTKQELK GLDIVRRDWC ELAKQAGNYV ISQILSDQPR DSIVENIQKK LTEIGENVTN GTVPITQYEI NKALTKDPQD YPDKKSLPHV HVALWINSQG GRKVKAGDTI SYVICQDGSN LSASQRAYAQ EQLQKQENLS IDTQYYLSQQ VHPVVARICE PIDGIDSALI AMWLGLDPSQ FRAHRHYQQD EENDALLGGP SQLTDEEKYR DCERFKFFCP KCGTENIYDN VFDGSGLQIE PGLKRCSKPE CDASPLDYVI QVHNKLLLDI RRYIKKYYSG WLVCEEKTCQ NRTRRLPLSF SRNGPICQAC SKATLRSEYP EKALYTQLCF YRFIFDWDYA LEKVVSEQER GHLKKKLFQE SENQYKKLKS TVDQVLSRSG YSEVNLSKLF QTLNTIK

UniProtKB: DNA polymerase alpha catalytic subunit

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Macromolecule #2: POLA2

MacromoleculeName: POLA2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
SequenceString: SNAMSVSAKS IAEELKVFDV NFEDEEVPEK MVELCTVHRL KEEDMVNEWM AFSTTRNLPL TVGNLNLLEH EVLNKKSARP RPSLKKEKHC GNRDFNTIQE LIEVETAEEN LLDSYATPAK GSQKRNLSTP EHPQSKRILS INRSPHVLFS PTSFSPSATP SQKYGSRTNR ...String:
SNAMSVSAKS IAEELKVFDV NFEDEEVPEK MVELCTVHRL KEEDMVNEWM AFSTTRNLPL TVGNLNLLEH EVLNKKSARP RPSLKKEKHC GNRDFNTIQE LIEVETAEEN LLDSYATPAK GSQKRNLSTP EHPQSKRILS INRSPHVLFS PTSFSPSATP SQKYGSRTNR GEVVTTYGEL QGTTWNGGSG SNTNVELFTS LDEPLTKMYK FMFQKLMDIR EVVSIKIEEL GASLKDHFQI DEFTSVSLPA QETVTVLGQI GCDSNGKLNS KSVILEGDRE HSAGMQVPVD LSELKDYSLF PGQVVIMEGT NSTGRRFVPT KLYEGVPLPF HQPSKEFEEC PQQMVITACG PFTTSDTITY DALKDLIDIV NRDRPDICIL LGPFLDAKHE QIENLQLTVT FEDVFKRCLK MIIEGTRPSG CHLVIVPSLR DVHHDPVYPQ PPFSCFEPAK EDKERVHFVA DPCTLSVNGV VIGMTSTDLL FHMGAEEISS SAGAPDRFSR ILRHILTQRS YYPLYPPNEE INIDYEALYS YTPMPVTPDV FIVPSELRYF IKDVTGCICI NPGRLTKGLV GGTYARFLVK SGAMGSEGKR STCISAQVVR V

UniProtKB: DNA polymerase alpha subunit B

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Macromolecule #3: PRIM2

MacromoleculeName: PRIM2 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
SequenceString: MLFSRDRKYR HNTRLTGDRK GDLYPSSLQF YQHPPTENIS LIEFETFAIE RLKLLKAVEN LGVSYVKNSE EYSKKLELEL RKLKFPYRPL HEEISDDVYD LRRKDHISHF ILRLAYCQSE DLRRWFIQQE MDLFKFRFGL LTKESVQEFL KLNDLQYVAI SEDEKNMHKE ...String:
MLFSRDRKYR HNTRLTGDRK GDLYPSSLQF YQHPPTENIS LIEFETFAIE RLKLLKAVEN LGVSYVKNSE EYSKKLELEL RKLKFPYRPL HEEISDDVYD LRRKDHISHF ILRLAYCQSE DLRRWFIQQE MDLFKFRFGL LTKESVQEFL KLNDLQYVAI SEDEKNMHKE DLMNSSFGLS LTKMEDTEFY KVPFQAALDL VRPRKVFLWR GFAFIPHKDI VSIVLNDFRA KLSKALALSA RSLPVVQSDE RLQPLLNHLS HSYIGQDFSS QSNTGKISLE QIDGFAAKSF PLCMRQLHKS LRENHHLRHG GRMQYGLFLK GIGLTLEQAL QFWRLEFTKG KVDSEKFDKV YAYSIRHNYG KEGKRTDYTP YSCMKVILSN PPSQGDYHGC PFRHSDPELL KQKLQSFKVP SSGINQILEL VKGMHYQLAC QKYFELTHSV DDCGFSLNHP NQYFAESQKL LTGSREIKKE QTARDSPAVT ASQLSSSSSS ASIPKSQSSA PEMEDLEQIF SEY

UniProtKB: DNA primase large subunit

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Macromolecule #4: PRIM1

MacromoleculeName: PRIM1 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
SequenceString: GPHMDLSVYD PASLPDVLPL YYRRLFPFYQ YFRWLNYGGV VKNYFQHREF SFTLKDDVYV RYQSFNNQSE LEKEMQKMCP YKIDIGAVYS HRPSLHNTVK SGTFQAQEKE LVFDIDMTDY DDVRRCCSSA DICPKCWTLM TIAVRILDRA LAEDFGFKHR LWVYSGRRGV ...String:
GPHMDLSVYD PASLPDVLPL YYRRLFPFYQ YFRWLNYGGV VKNYFQHREF SFTLKDDVYV RYQSFNNQSE LEKEMQKMCP YKIDIGAVYS HRPSLHNTVK SGTFQAQEKE LVFDIDMTDY DDVRRCCSSA DICPKCWTLM TIAVRILDRA LAEDFGFKHR LWVYSGRRGV HCWVCDDSAR KLSQAERSAV AEYLSVVKGG EETIKKVQLP ETIHPFIGKS LKMVERYFEK YALVDQDILE NKQCWDKVIA LVPEVARESL LREFSKARSS VERWDKLSSC LEATGKDFRR YSNIPKEIML QFCYPRLDVN VSKGLNHLLK SPFSVHPKTG RISVPIDCKK LDQFDPFSVP TISLICSELD NVSKKEEDED SAGEGEPEAK KRTRDYKRTS LAPYIKVFEQ FLDKLDQSRK GELLNKSDLK KEF

UniProtKB: DNA primase

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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

Concentration0.9 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 17820 / Average electron dose: 54.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 23007566
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 68842
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8g9f


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8v5o:
Tetramer core subcomplex (conformation 3) of Xenopus laevis DNA polymerase alpha-primase

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