2C2D

Efficient and High Fidelity Incorporation of dCTP Opposite 7,8- Dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA Polymerase Dpo4


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.57 Å
  • R-Value Free: 0.280 
  • R-Value Work: 0.240 
  • R-Value Observed: 0.240 

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Literature

Efficient and High Fidelity Incorporation of Dctp Opposite 7,8-Dihydro-8-Oxodeoxyguanosine by Sulfolobus Solfataricus DNA Polymerase Dpo4

Zang, H.Irimia, A.Choi, J.-Y.Angel, K.C.Loukachevitch, L.V.Egli, M.P Guengerich, F.

(2006) J Biol Chem 281: 2358

  • DOI: https://doi.org/10.1074/jbc.M510889200
  • Primary Citation of Related Structures:  
    2C22, 2C28, 2C2D, 2C2E, 2C2R

  • PubMed Abstract: 

    DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed >/=95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.


  • Organizational Affiliation

    Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, TN 37232-0146, USA.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA POLYMERASE IV358Saccharolobus solfataricus P2Mutation(s): 0 
EC: 2.7.7.7
UniProt
Find proteins for Q97W02 (Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2))
Explore Q97W02 
Go to UniProtKB:  Q97W02
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ97W02
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
5'-D(*GP*GP*GP*GP*GP*AP*AP*GP*GP*AP *TP*TP*C)-3'B [auth P]13synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
5'-D(*TP*CP*AP*C 8OGP*GP*AP*AP*TP*CP*CP *TP*TP*CP*CP*CP*CP*C)-3'C [auth T]18synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.57 Å
  • R-Value Free: 0.280 
  • R-Value Work: 0.240 
  • R-Value Observed: 0.240 
  • Space Group: P 21 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 97.507α = 90
b = 102.017β = 90
c = 53.093γ = 90
Software Package:
Software NamePurpose
CNSrefinement
XDSdata reduction
XDSdata scaling
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-11-29
    Type: Initial release
  • Version 1.1: 2015-11-11
    Changes: Derived calculations, Non-polymer description, Other, Refinement description, Source and taxonomy, Version format compliance
  • Version 1.2: 2023-12-13
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description