Yang Gao / Yanxiang Cui / Tara Fox / Shiqiang Lin / Huaibin Wang / Natalia de Val / Z Hong Zhou / Wei Yang /
PubMed Abstract
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 ...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.
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
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
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
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
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
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
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
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
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
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
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