5L1K

PostInsertion complex of Human DNA Polymerase Eta bypassing an O6-Methyl-2'-deoxyguanosine : dC site

  • Classification: Transferase/DNA
  • Organism(s): Homo sapiens, synthetic construct
  • Expression System: Escherichia coli
  • Mutation(s): No 

  • Deposited: 2016-07-29 Released: 2016-10-05 
  • Deposition Author(s): Patra, A., Egli, M.
  • Funding Organization(s): National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS), National Institutes of Health/National Cancer Institute (NIH/NCI)

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.82 Å
  • R-Value Free: 0.188 
  • R-Value Work: 0.153 
  • R-Value Observed: 0.154 

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Ligand Structure Quality Assessment 


This is version 1.5 of the entry. See complete history


Literature

Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase eta.

Patra, A.Zhang, Q.Guengerich, F.P.Egli, M.

(2016) J Biol Chem 291: 24304-24313

  • DOI: https://doi.org/10.1074/jbc.M116.755462
  • Primary Citation of Related Structures:  
    5L1I, 5L1J, 5L1K, 5L1L

  • PubMed Abstract: 

    O 6 -Methyl-2'-deoxyguanosine (O 6 -MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O 6 -MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O 6 -MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O 6 - MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O 6 -MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O 6 -MeG:dC or O 6 -MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η.


  • 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*GP*(6OG)P*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*C)-3')C [auth P]8synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.82 Å
  • R-Value Free: 0.188 
  • R-Value Work: 0.153 
  • R-Value Observed: 0.154 
  • Space Group: P 61
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 98.459α = 90
b = 98.459β = 90
c = 82.112γ = 120
Software Package:
Software NamePurpose
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing

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 StatesES010375
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)United StatesES010546
National Institutes of Health/National Cancer Institute (NIH/NCI)United StatesCA160032

Revision History  (Full details and data files)

  • Version 1.0: 2016-10-05
    Type: Initial release
  • Version 1.1: 2016-10-19
    Changes: Database references
  • Version 1.2: 2016-11-23
    Changes: Database references
  • Version 1.3: 2017-09-20
    Changes: Advisory, Author supporting evidence, Database references
  • Version 1.4: 2022-03-23
    Changes: Advisory, Author supporting evidence, Database references, Derived calculations
  • Version 1.5: 2023-10-04
    Changes: Data collection, Refinement description