5EWF

Ternary complex of human DNA polymerase eta inserting rCTP opposite an 8-Oxodeoxyguanosine Lesion


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.78 Å
  • R-Value Free: 0.216 
  • R-Value Work: 0.179 
  • R-Value Observed: 0.181 

Starting Model: experimental
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This is version 1.5 of the entry. See complete history


Literature

Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase eta.

Su, Y.Egli, M.Guengerich, F.P.

(2016) J Biol Chem 291: 3747-3756

  • DOI: https://doi.org/10.1074/jbc.M115.706226
  • Primary Citation of Related Structures:  
    5EWE, 5EWF, 5EWG

  • PubMed Abstract: 

    Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.


  • Organizational Affiliation

    From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase eta435Homo sapiensMutation(s): 0 
Gene Names: POLHRAD30RAD30AXPV
EC: 2.7.7.7
UniProt & NIH Common Fund Data Resources
Find proteins for Q9Y253 (Homo sapiens)
Explore Q9Y253 
Go to UniProtKB:  Q9Y253
PHAROS:  Q9Y253
GTEx:  ENSG00000170734 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9Y253
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(*CP*AP*TP*(8OG)P*AP*TP*GP*AP*CP*GP*CP*T)-3')B [auth T]12synthetic construct
Sequence Annotations
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  • Reference Sequence

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

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.78 Å
  • R-Value Free: 0.216 
  • R-Value Work: 0.179 
  • R-Value Observed: 0.181 
  • Space Group: P 61
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 99.124α = 90
b = 99.124β = 90
c = 81.384γ = 120
Software Package:
Software NamePurpose
PHENIXrefinement
Cootmodel building
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
ARPmodel building

Structure Validation

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Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)United StatesR01 ES010375
National Institutes of Health/National Cancer Institute (NIH/NCI)United StatesP01 CA160032

Revision History  (Full details and data files)

  • Version 1.0: 2016-01-13
    Type: Initial release
  • Version 1.1: 2016-01-20
    Changes: Database references
  • Version 1.2: 2016-03-02
    Changes: Database references
  • Version 1.3: 2017-09-13
    Changes: Author supporting evidence, Database references, Derived calculations
  • Version 1.4: 2019-12-04
    Changes: Author supporting evidence
  • Version 1.5: 2023-09-27
    Changes: Data collection, Database references, Derived calculations, Refinement description