4C8M

Binary complex of the large fragment of DNA polymerase I from Thermus Aquaticus with the aritificial base pair d5SICS-dNaM at the postinsertion site (sequence context 2)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.57 Å
  • R-Value Free: 0.207 
  • R-Value Work: 0.178 
  • R-Value Observed: 0.180 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Structural Insights Into DNA Replication without Hydrogen Bonds.

Betz, K.Malyshev, D.A.Lavergne, T.Welte, W.Diederichs, K.Romesberg, F.E.Marx, A.

(2013) J Am Chem Soc 135: 18637

  • DOI: https://doi.org/10.1021/ja409609j
  • Primary Citation of Related Structures:  
    4C8K, 4C8L, 4C8M, 4C8N, 4C8O, 4CCH

  • PubMed Abstract: 

    The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.


  • Organizational Affiliation

    Departments of Chemistry and Biology, Konstanz Research School Chemical Biology, Universität Konstanz , Universitätsstrasse 10, D-78464 Konstanz, Germany.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
LARGE FRAGMENT OF TAQ DNA POLYMERASE I540Thermus aquaticusMutation(s): 0 
EC: 2.7.7.7
UniProt
Find proteins for P19821 (Thermus aquaticus)
Explore P19821 
Go to UniProtKB:  P19821
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP19821
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
PRIMER, 5'-D(*GP*CP*CP*AP*CP*GP*GP*CP*GP*CP*BMNP)-3'11synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
TEMPLATE, 5'-D(*TP*TP*CP* LHOP*GP*CP*GP*CP*CP*GP*TP*GP*GP*CP)-3'14synthetic construct
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SO4
Query on SO4

Download Ideal Coordinates CCD File 
D [auth A],
E [auth A],
G [auth A]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
GOL
Query on GOL

Download Ideal Coordinates CCD File 
F [auth A],
H [auth A],
I [auth A],
J [auth A],
M [auth C]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
MG
Query on MG

Download Ideal Coordinates CCD File 
K [auth B],
L [auth C]
MAGNESIUM ION
Mg
JLVVSXFLKOJNIY-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.57 Å
  • R-Value Free: 0.207 
  • R-Value Work: 0.178 
  • R-Value Observed: 0.180 
  • Space Group: C 2 2 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 65.374α = 90
b = 101.456β = 90
c = 204.543γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
XDSdata reduction
XDSdata scaling
PHENIXphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2013-12-11
    Type: Initial release
  • Version 1.1: 2013-12-25
    Changes: Atomic model, Database references
  • Version 1.2: 2017-08-23
    Changes: Data collection