5VS2

Human DNA polymerase beta pre-catalytic 8-oxoG:dA extension complex with dTTP bound in Watson-Crick conformation

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.33 Å
  • R-Value Free: 0.250 
  • R-Value Work: 0.187 
  • R-Value Observed: 0.190 

Starting Model: experimental
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This is version 1.4 of the entry. See complete history. 


Literature

Time-Dependent Extension from an 8-Oxoguanine Lesion by Human DNA Polymerase Beta.

Reed, A.J., Suo, Z.

(2017) J Am Chem Soc 139: 9684-9690

  • DOI: https://doi.org/10.1021/jacs.7b05048
  • Primary Citation of Related Structures:  
    5VRW, 5VRX, 5VRY, 5VRZ, 5VS0, 5VS1, 5VS2, 5VS3, 5VS4

  • PubMed Abstract: 

    The oxidative DNA lesion 7,8-dihydro-2'-deoxyguanine (8-oxoG) often occurs in double-stranded DNA and poses a threat to genomic integrity due to the ability of 8-oxoG to form stable Watson-Crick base pairs with deoxycytidine (8-oxoG:dC) and Hoogsteen base pairs with deoxyadenosine (8-oxoG:dA). In humans, short-patch base excision repair of 8-oxoG:dA base pairs requires human DNA polymerase β (hPolβ) to bypass 8-oxoG. Previously, we have shown hPolβ-catalyzed 8-oxoG bypass to exhibit low fidelity and identified a unique stacking interaction between the newly incorporated nucleotide (dCMP or dAMP) and the templating 8-oxoG. The effect of this stacking on the ability of hPolβ to extend from 8-oxoG during long-patch base excision repair was unknown. Here we report pre-steady-state kinetics and time-dependent crystal structures to demonstrate that extension from both 8-oxoG:dC and 8-oxoG:dA base pairs is 18- to 580-fold less efficient compared to 8-oxoG bypass and that extension from 8-oxoG:dC over 8-oxoG:dA is favored by 15-fold. The overall decrease in efficiency of extension relative to 8-oxoG bypass is due to an alternative nucleotide binding conformation in the precatalytic ternary structures (hPolβ·DNA·dNTP) for both extension contexts, wherein the incoming nucleotide is bound in either the canonical Watson-Crick base pair or a nonplanar base pair. In addition, the decreased stability of the ternary complex of 8-oxoG:dA extension results in further loss of efficiency when compared to 8-oxoG:dC extension. Therefore, we hypothesize that the inefficient extension from 8-oxoG:dA serves as a newly discovered fidelity checkpoint during base excision repair.

    • Organizational Affiliation: 

    Department of Chemistry and Biochemistry, The Ohio State Biochemistry Program, The Ohio State University , Columbus, Ohio 43210, United States.


Macromolecules

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Entity ID: 4
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase betaD [auth A]341Homo sapiensMutation(s): 0 
Gene Names: POLB
EC: 2.7.7.7 (PDB Primary Data), 4.2.99 (PDB Primary Data), 4.2.99.18 (UniProt)
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*AP*(8OG)P*GP*CP*GP*CP*AP*TP*CP*AP*G)-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(*CP*TP*GP*AP*TP*GP*CP*GP*CP*A)-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
Small Molecules
Binding Affinity Annotations 
IDSourceBinding Affinity
TTP BindingDB:  5VS2 IC50: 1400 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.33 Å
  • R-Value Free: 0.250 
  • R-Value Work: 0.187 
  • R-Value Observed: 0.190 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 50.94α = 90
b = 80.49β = 107.28
c = 55.66γ = 90
Software Package:
Software NamePurpose
Aimlessdata scaling
REFMACrefinement
PDB_EXTRACTdata extraction
iMOSFLMdata reduction
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


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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 StatesES024585
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)United StatesES026821

Revision History  (Full details and data files)

  • Version 1.0: 2017-07-19
    Type: Initial release
  • Version 1.1: 2017-07-26
    Changes: Database references
  • Version 1.2: 2017-11-22
    Changes: Refinement description
  • Version 1.3: 2022-03-23
    Changes: Author supporting evidence, Database references, Derived calculations
  • Version 1.4: 2023-10-04
    Changes: Data collection, Refinement description