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Structure paper

TitleStructures and operating principles of the replisome.
Journal, issue, pagesScience, Vol. 363, Issue 6429, Year 2019
Publish dateFeb 22, 2019
AuthorsYang Gao / Yanxiang Cui / Tara Fox / Shiqiang Lin / Huaibin Wang / Natalia de Val / Z Hong Zhou / Wei Yang /
PubMed AbstractVisualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model ...Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo-electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.
External linksScience / PubMed:30679383 / PubMed Central
MethodsEM (single particle)
Resolution3.2 - 13.8 Å
Structure data

0357

6n7i

EMDB-0357, PDB-6n7i:
Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (gp4(5)-DNA)
Method: EM (single particle) / Resolution: 3.2 Å

0359

6n7n

EMDB-0359, PDB-6n7n:
Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (form I)
Method: EM (single particle) / Resolution: 3.5 Å

0362

6n7s

EMDB-0362, PDB-6n7s:
Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (form II)
Method: EM (single particle) / Resolution: 4.6 Å

0363

6n7t

EMDB-0363, PDB-6n7t:
Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (form III)
Method: EM (single particle) / Resolution: 3.9 Å

0364

6n7v

EMDB-0364, PDB-6n7v:
Structure of bacteriophage T7 gp4 (helicase-primase, E343Q mutant) in complex with ssDNA, dTTP, AC dinucleotide, and CTP (from multiple lead complexes)
Method: EM (single particle) / Resolution: 3.8 Å

0365

6n7w

EMDB-0365, PDB-6n7w:
Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx in complex with a DNA fork and incoming dTTP (from multiple lead complexes)
Method: EM (single particle) / Resolution: 4.5 Å

0379

6n9u

EMDB-0379, PDB-6n9u:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A) interacting with primase domains of two gp4 subunits bound to an RNA/DNA hybrid and dTTP (from LagS1)
Method: EM (single particle) / Resolution: 3.7 Å

0380

6n9v

EMDB-0380, PDB-6n9v:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A) and gp4 (helicase/primase) bound to DNA including RNA/DNA hybrid, and an incoming dTTP (LagS1)
Method: EM (single particle) / Resolution: 4.0 Å

0381

6n9w

EMDB-0381, PDB-6n9w:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A) and gp4 (helicase/primase) bound to DNA including RNA/DNA hybrid, and an incoming dTTP (LagS2)
Method: EM (single particle) / Resolution: 4.0 Å

0382

6n9x

EMDB-0382, PDB-6n9x:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A) and gp4 (helicase/primase) bound to DNA including RNA/DNA hybrid, and an incoming dTTP (LagS3)
Method: EM (single particle) / Resolution: 4.1 Å

EMDB-0386:
Structure of two bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A )/Trx interacting with primase domains, one Pol with RNA/DNA hybrid, and dTTP interacting and the second Pol in apo form (LagS4)
Method: EM (single particle) / Resolution: 8.6 Å

EMDB-0387:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A)/Trx interacting with primase domains of two gp4 subunits (E and F), with gp4 helicase bound to a DNA fork and dTTP (LagL1)
Method: EM (single particle) / Resolution: 6.6 Å

EMDB-0388:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A)/Trx interacting with primase domains of two gp4 subunits (C and D), with gp4 helicase bound to a DNA fork and dTTP (LagL2)
Method: EM (single particle) / Resolution: 7.1 Å

EMDB-0389:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A)/Trx interacting with primase domains of two gp4 subunits (B and C), with gp4 helicase bound to a DNA fork and dTTP (LagL3)
Method: EM (single particle) / Resolution: 7.8 Å

EMDB-0390:
Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A)/Trx interacting with primase domains of two gp4 subunits (A and B), with gp4 helicase bound to a DNA fork and dTTP (LagL4)
Method: EM (single particle) / Resolution: 8.3 Å

EMDB-0391:
Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx and of gp4 (E343Q) bound to a DNA fork (Lead1)
Method: EM (single particle) / Resolution: 11.2 Å

EMDB-0392:
Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx and of gp4 (E343Q) bound to a DNA fork (Lead2)
Method: EM (single particle) / Resolution: 13.3 Å

EMDB-0393:
Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx and of T7 gp4 (E343Q) bound to a DNA fork, and dTTP (Lead3)
Method: EM (single particle) / Resolution: 11.8 Å

EMDB-0394:
Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx and of gp4 (E343Q) bound to a DNA fork (Lead4)
Method: EM (single particle) / Resolution: 9.6 Å

EMDB-0395:
Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx and of gp4 (E343Q) bound to a DNA fork (Lead5)
Method: EM (single particle) / Resolution: 13.8 Å

Chemicals

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

ChemComp-MG:
Unknown entry

ChemComp-HOH:
WATER

ChemComp-ZN:
Unknown entry

Source
  • enterobacteria phage t7 (virus)
  • escherichia coli (E. coli)
KeywordsHYDROLASE / TRANSFERASE/DNA / helicase / ATPase / hexamer / DNA replication / TRANSFERASE-DNA complex / DNA polymerase / replisome / primase

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