5WNZ

DNA polymerase beta substrate complex with incoming 5-FodCTP


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.254 
  • R-Value Work: 0.193 
  • R-Value Observed: 0.199 

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Literature

Structures of a DNA Polymerase Inserting Therapeutic Nucleotide Analogues.

Schaich, M.A.Smith, M.R.Cloud, A.S.Holloran, S.M.Freudenthal, B.D.

(2017) Chem Res Toxicol 30: 1993-2001

  • DOI: https://doi.org/10.1021/acs.chemrestox.7b00173
  • Primary Citation of Related Structures:  
    5WNX, 5WNY, 5WNZ, 5WO0

  • PubMed Abstract: 

    Members of the nucleoside analogue class of cancer therapeutics compete with canonical nucleotides to disrupt numerous cellular processes, including nucleotide homeostasis, DNA and RNA synthesis, and nucleotide metabolism. Nucleoside analogues are triphosphorylated and subsequently inserted into genomic DNA, contributing to the efficacy of therapeutic nucleosides in multiple ways. In some cases, the altered base acts as a mutagen, altering the DNA sequence to promote cellular death; in others, insertion of the altered nucleotide triggers DNA repair pathways, which produce lethal levels of cytotoxic intermediates such as single and double stranded DNA breaks. As a prerequisite to many of these biological outcomes, the modified nucleotide must be accommodated in the DNA polymerase active site during nucleotide insertion. Currently, the molecular contacts that mediate DNA polymerase insertion of modified nucleotides remain unknown for multiple therapeutic compounds, despite decades of clinical use. To determine how modified bases are inserted into duplex DNA, we used mammalian DNA polymerase β (pol β) to visualize the structural conformations of four therapeutically relevant modified nucleotides, 6-thio-2'-deoxyguanosine-5'-triphosphate (6-TdGTP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (5-FdUTP), 5-formyl-deoxycytosine-5'-triphosphate (5-FodCTP), and 5-formyl-deoxyuridine-5'-triphosphate (5-FodUTP). Together, the structures reveal a pattern in which the modified nucleotides utilize Watson-Crick base pairing interactions similar to that of unmodified nucleotides. The nucleotide modifications were consistently positioned in the major groove of duplex DNA, accommodated by an open cavity in pol β. These results provide novel information for the rational design of new therapeutic nucleoside analogues and a greater understanding of how modified nucleotides are tolerated by polymerases.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.


Macromolecules

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Entity ID: 4
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase betaD [auth A]335Homo sapiensMutation(s): 0 
Gene Names: POLB
EC: 2.7.7.7 (PDB Primary Data), 4.2.99 (PDB Primary Data)
UniProt & NIH Common Fund Data Resources
Find proteins for P06746 (Homo sapiens)
Explore P06746 
Go to UniProtKB:  P06746
PHAROS:  P06746
GTEx:  ENSG00000070501 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP06746
Sequence Annotations
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  • Reference Sequence

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Entity ID: 1
MoleculeChains LengthOrganismImage
DNA (5'-D(*CP*CP*GP*AP*CP*GP*GP*CP*GP*CP*AP*TP*CP*AP*GP*C)-3')A [auth T]16synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(*GP*CP*TP*GP*AP*TP*GP*CP*GP*C)-3')B [auth P]10synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
DNA (5'-D(P*GP*TP*CP*GP*G)-3')C [auth D]5synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.254 
  • R-Value Work: 0.193 
  • R-Value Observed: 0.199 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 50.607α = 90
b = 79.627β = 107.24
c = 55.624γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-3000data reduction
HKL-3000data scaling
PHENIXphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)United StatesES024432

Revision History  (Full details and data files)

  • Version 1.0: 2017-09-13
    Type: Initial release
  • Version 1.1: 2017-11-29
    Changes: Author supporting evidence, Database references
  • Version 1.2: 2019-12-18
    Changes: Author supporting evidence
  • Version 1.3: 2023-10-04
    Changes: Data collection, Database references, Derived calculations, Refinement description