5M1S

Cryo-EM structure of the E. coli replicative DNA polymerase-clamp-exonuclase-theta complex bound to DNA in the editing mode


Experimental Data Snapshot

  • Method: ELECTRON MICROSCOPY
  • Resolution: 6.70 Å
  • Aggregation State: PARTICLE 
  • Reconstruction Method: SINGLE PARTICLE 

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This is version 1.3 of the entry. See complete history


Literature

Self-correcting mismatches during high-fidelity DNA replication.

Fernandez-Leiro, R.Conrad, J.Yang, J.C.Freund, S.M.Scheres, S.H.Lamers, M.H.

(2017) Nat Struct Mol Biol 24: 140-143

  • DOI: https://doi.org/10.1038/nsmb.3348
  • Primary Citation of Related Structures:  
    5M1S

  • PubMed Abstract: 

    Faithful DNA replication is essential to all forms of life and depends on the action of 3'-5' exonucleases that remove misincorporated nucleotides from the newly synthesized strand. However, how the DNA is transferred from the polymerase to the exonuclease active site is not known. Here we present the cryo-EM structure of the editing mode of the catalytic core of the Escherichia coli replisome, revealing a dramatic distortion of the DNA whereby the polymerase thumb domain acts as a wedge that separates the two DNA strands. Importantly, NMR analysis of the DNA substrate shows that the presence of a mismatch increases the fraying of the DNA, thus enabling it to reach the exonuclease active site. Therefore the mismatch corrects itself, whereas the exonuclease subunit plays a passive role. Hence, our work provides unique insights into high-fidelity replication and establishes a new paradigm for the correction of misincorporated nucleotides.


  • Organizational Affiliation

    MRC laboratory of Molecular Biology, Cambridge, UK.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase III subunit alpha927Escherichia coli K-12Mutation(s): 2 
Gene Names: dnaEpolCb0184JW0179
EC: 2.7.7.7
UniProt
Find proteins for P10443 (Escherichia coli (strain K12))
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Go to UniProtKB:  P10443
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UniProt GroupP10443
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  • Reference Sequence
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase III subunit beta
B, C
366Escherichia coli K-12Mutation(s): 0 
Gene Names: dnaNb3701JW3678
EC: 2.7.7.7
UniProt
Find proteins for P0A988 (Escherichia coli (strain K12))
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Go to UniProtKB:  P0A988
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UniProt GroupP0A988
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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase III subunit epsilon243Escherichia coli K-12Mutation(s): 4 
Gene Names: dnaQmutDb0215JW0205
EC: 2.7.7.7
UniProt
Find proteins for P03007 (Escherichia coli (strain K12))
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UniProt GroupP03007
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Entity ID: 6
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase III subunit thetaG [auth F]56Escherichia coli K-12Mutation(s): 0 
Gene Names: holEb1842JW1831
EC: 2.7.7.7
UniProt
Find proteins for P0ABS8 (Escherichia coli (strain K12))
Explore P0ABS8 
Go to UniProtKB:  P0ABS8
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UniProt GroupP0ABS8
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Entity ID: 4
MoleculeChains LengthOrganismImage
DNA Primer StrandE [auth P]17synthetic construct
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Entity ID: 5
MoleculeChains LengthOrganismImage
DNA Template StrandF [auth T]22synthetic construct
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: ELECTRON MICROSCOPY
  • Resolution: 6.70 Å
  • Aggregation State: PARTICLE 
  • Reconstruction Method: SINGLE PARTICLE 
EM Software:
TaskSoftware PackageVersion
RECONSTRUCTIONRELION2
MODEL REFINEMENTREFMAC5.8
MODEL REFINEMENTLIBG

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2017-01-18
    Type: Initial release
  • Version 1.1: 2017-02-15
    Changes: Database references
  • Version 1.2: 2017-08-30
    Changes: Data collection, Derived calculations, Experimental preparation
  • Version 1.3: 2018-10-24
    Changes: Advisory, Data collection, Derived calculations