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- EMDB-29347: Cryo-EM structure of S. cerevisiae DNA polymerase alpha-primase c... -

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Entry
Database: EMDB / ID: EMD-29347
TitleCryo-EM structure of S. cerevisiae DNA polymerase alpha-primase complex bound to a template DNA
Map data
Sample
  • Complex: DNA polymerase alpha/primase complex
    • Protein or peptide: DNA primase
    • Protein or peptide: DNA primase large subunit
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase alpha subunit B
  • Ligand: IRON/SULFUR CLUSTER
KeywordsDNA polymerase / primase / DNA replication / DNA BINDING PROTEIN
Function / homology
Function and homology information


alpha DNA polymerase:primase complex / DNA primase activity / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / leading strand elongation / DNA replication origin binding / DNA replication initiation / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / single-stranded DNA binding ...alpha DNA polymerase:primase complex / DNA primase activity / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / leading strand elongation / DNA replication origin binding / DNA replication initiation / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / DNA binding / metal ion binding
Similarity search - Function
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 ...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 / DNA primase / DNA polymerase alpha subunit B
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.6 Å
AuthorsYuan Z / Georgescu R / Li H / O'Donnell M
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115809 United States
Howard Hughes Medical Institute (HHMI)M.E.O. United States
CitationJournal: bioRxiv / Year: 2023
Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase.
Authors: Zuanning Yuan / Roxana Georgescu / Huilin Li / Michael E O'Donnell /
Abstract: The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, ...The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.
History
DepositionDec 30, 2022-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29347.png

Downloads & links

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Map

FileDownload / File: emd_29347.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.41
Minimum - Maximum-0.2414525 - 1.5159281
Average (Standard dev.)0.017085338 (±0.08250145)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29347_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29347_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : DNA polymerase alpha/primase complex

EntireName: DNA polymerase alpha/primase complex
Components
  • Complex: DNA polymerase alpha/primase complex
    • Protein or peptide: DNA primase
    • Protein or peptide: DNA primase large subunit
    • Protein or peptide: DNA polymerase
    • Protein or peptide: DNA polymerase alpha subunit B
  • Ligand: IRON/SULFUR CLUSTER

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Supramolecule #1: DNA polymerase alpha/primase complex

SupramoleculeName: DNA polymerase alpha/primase complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#3, #1, #4
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 167.027766 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSSKSEKLEK LRKLQAARNG TSIDDYEGDE SDGDRIYDEI DEKEYRARKR QELLHDDFVV DDDGVGYVDR GVEEDWREVD NSSSDEDTG NLASKDSKRK KNIKREKDHQ ITDMLRTQHS KSTLLAHAKK SQKKSIPIDN FDDILGEFES GEVEKPNILL P SKLRENLN ...String:
MSSKSEKLEK LRKLQAARNG TSIDDYEGDE SDGDRIYDEI DEKEYRARKR QELLHDDFVV DDDGVGYVDR GVEEDWREVD NSSSDEDTG NLASKDSKRK KNIKREKDHQ ITDMLRTQHS KSTLLAHAKK SQKKSIPIDN FDDILGEFES GEVEKPNILL P SKLRENLN SSPTSEFKSS IKRVNGNDES SHDAGISKKV KIDPDSSTDK YLEIESSPLK LQSRKLRYAN DVQDLLDDVE NS PVVATKR QNVLQDTLLA NPPSAQSLAD EEDDEDSDED IILKRRTMRS VTTTRRVNID SRSNPSTSPF VTAPGTPIGI KGL TPSKSL QSNTDVATLA VNVKKEDVVD PETDTFQMFW LDYCEVNNTL ILFGKVKLKD DNCVSAMVQI NGLCRELFFL PREG KTPTD IHEEIIPLLM DKYGLDNIRA KPQKMKYSFE LPDIPSESDY LKVLLPYQTP KSSRDTIPSD LSSDTFYHVF GGNSN IFES FVIQNRIMGP CWLDIKGADF NSIRNASHCA VEVSVDKPQN ITPTTTKTMP NLRCLSLSIQ TLMNPKENKQ EIVSIT LSA YRNISLDSPI PENIKPDDLC TLVRPPQSTS FPLGLAALAK QKLPGRVRLF NNEKAMLSCF CAMLKVEDPD VIIGHRL QN VYLDVLAHRM HDLNIPTFSS IGRRLRRTWP EKFGRGNSNM NHFFISDICS GRLICDIANE MGQSLTPKCQ SWDLSEMY Q VTCEKEHKPL DIDYQNPQYQ NDVNSMTMAL QENITNCMIS AEVSYRIQLL TLTKQLTNLA GNAWAQTLGG TRAGRNEYI LLHEFSRNGF IVPDKEGNRS RAQKQRQNEE NADAPVNSKK AKYQGGLVFE PEKGLHKNYV LVMDFNSLYP SIIQEFNICF TTVDRNKED IDELPSVPPS EVDQGVLPRL LANLVDRRRE VKKVMKTETD PHKRVQCDIR QQALKLTANS MYGCLGYVNS R FYAKPLAM LVTNKGREIL MNTRQLAESM NLLVVYGDTD SVMIDTGCDN YADAIKIGLG FKRLVNERYR LLEIDIDNVF KK LLLHAKK KYAALTVNLD KNGNGTTVLE VKGLDMKRRE FCPLSRDVSI HVLNTILSDK DPEEALQEVY DYLEDIRIKV ETN NIRIDK YKINMKLSKD PKAYPGGKNM PAVQVALRMR KAGRVVKAGS VITFVITKQD EIDNAADTPA LSVAERAHAL NEVM IKSNN LIPDPQYYLE KQIFAPVERL LERIDSFNVV RLSEALGLDS KKYFRREGGN NNGEDINNLQ PLETTITDVE RFKDT VTLE LSCPSCDKRF PFGGIVSSNY YRVSYNGLQC KHCEQLFTPL QLTSQIEHSI RAHISLYYAG WLQCDDSTCG IVTRQV SVF GKRCLNDGCT GVMRYKYSDK QLYNQLLYFD SLFDCEKNKK QELKPIYLPD DLDYPKEQLT ESSIKALTEQ NRELMET GR SVVQKYLNDC GRRYVDMTSI FDFMLN

UniProtKB: DNA polymerase

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Macromolecule #2: DNA primase

MacromoleculeName: DNA primase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 47.760367 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MTNSVKTNGP SSSDMEYYYK SLYPFKHIFN WLNHSPKPSR DMINREFAMA FRSGAYKRYN SFNSVQDFKA QIEKANPDRF EIGAIYNKP PRERDTLLKS ELKALEKELV FDIDMDDYDA FRTCCSGAQV CSKCWKFISL AMKITNTALR EDFGYKDFIW V FSGRRGAH ...String:
MTNSVKTNGP SSSDMEYYYK SLYPFKHIFN WLNHSPKPSR DMINREFAMA FRSGAYKRYN SFNSVQDFKA QIEKANPDRF EIGAIYNKP PRERDTLLKS ELKALEKELV FDIDMDDYDA FRTCCSGAQV CSKCWKFISL AMKITNTALR EDFGYKDFIW V FSGRRGAH CWVSDKRARA LTDVQRRNVL DYVNVIRDRN TDKRLALKRP YHPHLARSLE QLKPFFVSIM LEEQNPWEDD QH AIQTLLP ALYDKQLIDS LKKYWLDNPR RSSKEKWNDI DQIATSLFKG PKQDSHIIKL RECKEDLVLM TLYPKLDVEV TKQ TIHLLK APFCIHPATG NVCVPIDESF APEKAPKLID LQTEMEKNND VSLTALQPFI NQFQAYVSSL LKNELGSVKR ERED DDEPA SLDF

UniProtKB: DNA primase

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Macromolecule #3: DNA primase large subunit

MacromoleculeName: DNA primase large subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 62.305457 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MFRQSKRRIA SRKNFSSYDD IVKSELDVGN TNAANQIILS SSSSEEEKKL YARLYESKLS FYDLPPQGEI TLEQFEIWAI DRLKILLEI ESCLSRNKSI KEIETIIKPQ FQKLLPFNTE SLEDRKKDYY SHFILRLCFC RSKELREKFV RAETFLFKIR F NMLTSTDQ ...String:
MFRQSKRRIA SRKNFSSYDD IVKSELDVGN TNAANQIILS SSSSEEEKKL YARLYESKLS FYDLPPQGEI TLEQFEIWAI DRLKILLEI ESCLSRNKSI KEIETIIKPQ FQKLLPFNTE SLEDRKKDYY SHFILRLCFC RSKELREKFV RAETFLFKIR F NMLTSTDQ TKFVQSLDLP LLQFISNEEK AELSHQLYQT VSASLQFQLN LNEEHQRKQY FQQEKFIKLP FENVIELVGN RL VFLKDGY AYLPQFQQLN LLSNEFASKL NQELIKTYQY LPRLNEDDRL LPILNHLSSG YTIADFNQQK ANQFSENVDD EIN AQSVWS EEISSNYPLC IKNLMEGLKK NHHLRYYGRQ QLSLFLKGIG LSADEALKFW SEAFTNNGNM TMEKFNKEYR YSFR HNYGL EGNRINYKPW DCHTILSKPR PGRGDYHGCP FRDWSHERLS AELRSMKLTQ AQIISVLDSC QKGEYTIACT KVFEM THNS ASADLEIGEQ THIAHPNLYF ERSRQLQKKQ QKLEKEKLFN NGNH

UniProtKB: DNA primase large subunit

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Macromolecule #4: DNA polymerase alpha subunit B

MacromoleculeName: DNA polymerase alpha subunit B / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 78.865938 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSGSIDVITH FGPDADKPEI ITALENLTKL HALSVEDLYI KWEQFSNQRR QTHTDLTSKN IDEFKQFLQL QMEKRANQIS SSSKVNTST KKPVIKKSLN SSPLFGLSIP KTPTLKKRKL HGPFSLSDSK QTYNVGSEAE TNEKGNSSLK LEFTPGMAED A VGDSAPLS ...String:
MSGSIDVITH FGPDADKPEI ITALENLTKL HALSVEDLYI KWEQFSNQRR QTHTDLTSKN IDEFKQFLQL QMEKRANQIS SSSKVNTST KKPVIKKSLN SSPLFGLSIP KTPTLKKRKL HGPFSLSDSK QTYNVGSEAE TNEKGNSSLK LEFTPGMAED A VGDSAPLS HAKSSDAKTP GSSTFQTPTT NTPTTSRQNV PAGEILDSLN PENIEISSGN PNVGLLSTEE PSYNQVKVEP FY DAKKYKF RTMRQNLQEA SDVLDDQIES FTKIIQNHYK LSPNDFADPT IQSQSEIYAV GRIVPDSPTY DKFLNPESLS LET SRMGGV GRRVRLDLSQ VNELSFFLGQ IVAFKGKNAN GDYFTVNSIL PLPYPNSPVS TSQELQEFQA NLEGSSLKVI VTCG PYFAN DNFSLELLQE FIDSINNEVK PHVLIMFGPF IDITHPLIAS GKLPNFPQFK TQPKTLDELF LKLFTPILKT ISPHI QTVL IPSTKDAISN HAAYPQASLI RKALQLPKRN FKCMANPSSF QINEIYFGCS NVDTFKDLKE VIKGGTTSSR YRLDRV SEH ILQQRRYYPI FPGSIRTRIK PKDVSTKKET NDMESKEEKV YEHISGADLD VSYLGLTEFV GGFSPDIMII PSELQHF AR VVQNVVVINP GRFIRATGNR GSYAQITVQC PDLEDGKLTL VEGEEPVYLH NVWKRARVDL IAS

UniProtKB: DNA polymerase alpha subunit B

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 176443
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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