3TAG

5-fluorocytosine paired with dAMP in RB69 gp43


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.95 Å
  • R-Value Free: 0.284 
  • R-Value Work: 0.239 
  • R-Value Observed: 0.243 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

The miscoding potential of 5-hydroxycytosine arises due to template instability in the replicative polymerase active site.

Zahn, K.E.Averill, A.Wallace, S.S.Doublie, S.

(2011) Biochemistry 50: 10350-10358

  • DOI: https://doi.org/10.1021/bi201219s
  • Primary Citation of Related Structures:  
    3TAB, 3TAE, 3TAF, 3TAG

  • PubMed Abstract: 

    5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C → T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, a high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC ~5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.


  • Organizational Affiliation

    Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, United States.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA-directed DNA polymerase
A, B, C, D
906Escherichia phage RB69Mutation(s): 2 
Gene Names: 43
EC: 2.7.7.7 (PDB Primary Data), 3.1.11 (PDB Primary Data)
UniProt
Find proteins for Q38087 (Escherichia phage RB69)
Explore Q38087 
Go to UniProtKB:  Q38087
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ38087
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(*CP*CP*(C37)P*GP*GP*TP*AP*TP*GP*AP*CP*AP*GP*CP*CP*GP*CP*G)-3')E,
G [auth I],
I [auth G],
K
18synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
DNA (5'-D(*GP*CP*GP*GP*CP*TP*GP*TP*CP*AP*TP*AP*CP*CP*A)-3')F,
H [auth J],
J [auth H],
L
15synthetic construct
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SO4
Query on SO4

Download Ideal Coordinates CCD File 
M [auth A]SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.95 Å
  • R-Value Free: 0.284 
  • R-Value Work: 0.239 
  • R-Value Observed: 0.243 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 132.771α = 90
b = 123.028β = 96.39
c = 165.184γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
CNSrefinement
Blu-Icedata collection
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing

Structure Validation

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Entry History 

Deposition Data

  • Released Date: 2011-11-09 
  • Deposition Author(s): Zahn, K.E.

Revision History  (Full details and data files)

  • Version 1.0: 2011-11-09
    Type: Initial release
  • Version 1.1: 2012-01-11
    Changes: Database references
  • Version 1.2: 2019-07-17
    Changes: Data collection, Derived calculations, Refinement description
  • Version 1.3: 2024-02-28
    Changes: Data collection, Database references, Derived calculations, Source and taxonomy, Structure summary