2W9B

Binary complex of Dpo4 bound to N2,N2-dimethyl-deoxyguanosine modified DNA


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.28 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.239 
  • R-Value Observed: 0.239 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Structure-Function Relationships in Miscoding by Sulfolobus Solfataricus DNA Polymerase Dpo4: Guanine N2,N2-Dimethyl Substitution Produces Inactive and Miscoding Polymerase Complexes.

Zhang, H.Eoff, R.L.Kosekov, I.D.Rizzo, C.J.Egli, M.Guengerich, F.P.

(2009) J Biol Chem 284: 17687

  • DOI: https://doi.org/10.1074/jbc.M109014274
  • Primary Citation of Related Structures:  
    2W9A, 2W9B, 2W9C

  • PubMed Abstract: 

    Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N2,N2-Me2G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3'-terminal dideoxycytosine (Cdd) opposite template N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G adducts.


  • Organizational Affiliation

    Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 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 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
Expand
  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
5'-D(*GP*GP*GP*GP*GP*AP*AP*GP*GP*AP *TP*TP*CP*DOCP)-3'
C, D
14N/A
Sequence Annotations
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  • Reference Sequence

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Entity ID: 4
MoleculeChains LengthOrganismImage
5'-D(*TP*CP*AP*TP*M2GP*GP*AP*AP*TP*CP*CP *TP*TP*CP*CP*CP*CP*C)-3'
E, F
18N/A
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.28 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.239 
  • R-Value Observed: 0.239 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 51.794α = 90
b = 101.776β = 91.95
c = 97.268γ = 90
Software Package:
Software NamePurpose
CNSrefinement
XDSdata reduction
XDSdata scaling
MOLREPphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2009-05-12
    Type: Initial release
  • Version 1.1: 2011-05-08
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2023-12-13
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description