5CWR

Crystal Structure of human DNA polymerase lambda L431A mutant in complex with a one nucleotide DNA gap and dCTP


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.234 
  • R-Value Work: 0.202 
  • R-Value Observed: 0.203 

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Literature

Structural Mechanism for the Fidelity Modulation of DNA Polymerase lambda

Liu, M.S.Tsai, H.Y.Liu, X.X.Ho, M.C.Wu, W.J.Tsai, M.D.

(2016) J Am Chem Soc 138: 2389-2398

  • DOI: https://doi.org/10.1021/jacs.5b13368
  • Primary Citation of Related Structures:  
    4XQ8, 4XRH, 5CA7, 5CB1, 5CHG, 5CJ7, 5CP2, 5CR0, 5CWR, 5DDM, 5DDY, 5DKW

  • PubMed Abstract: 

    The mechanism of DNA polymerase (pol) fidelity is of fundamental importance in chemistry and biology. While high-fidelity pols have been well studied, much less is known about how some pols achieve medium or low fidelity with functional importance. Here we examine how human DNA polymerase λ (Pol λ) achieves medium fidelity by determining 12 crystal structures and performing pre-steady-state kinetic analyses. We showed that apo-Pol λ exists in the closed conformation, unprecedentedly with a preformed MgdNTP binding pocket, and binds MgdNTP readily in the active conformation in the absence of DNA. Since prebinding of MgdNTP could lead to very low fidelity as shown previously, it is attenuated in Pol λ by a hydrophobic core including Leu431, Ile492, and the Tyr505/Phe506 motif. We then predicted and demonstrated that L431A mutation enhances MgdNTP prebinding and lowers the fidelity. We also hypothesized that the MgdNTP-prebinding ability could stabilize a mismatched ternary complex and destabilize a matched ternary complex, and provided evidence with structures in both forms. Our results demonstrate that, while high-fidelity pols follow a common paradigm, Pol λ has developed specific conformations and mechanisms for its medium fidelity. Structural comparison with other pols also suggests that different pols likely utilize different conformational changes and microscopic mechanisms to achieve their catalytic functions with varying fidelities.


  • Organizational Affiliation

    Institute of Biochemical Sciences, National Taiwan University , Taipei 106, Taiwan.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase lambdaA,
D [auth B]
326Homo sapiensMutation(s): 1 
Gene Names: POLL
EC: 2.7.7.7
UniProt & NIH Common Fund Data Resources
Find proteins for Q9UGP5 (Homo sapiens)
Explore Q9UGP5 
Go to UniProtKB:  Q9UGP5
PHAROS:  Q9UGP5
GTEx:  ENSG00000166169 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9UGP5
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(*CP*GP*GP*CP*GP*GP*TP*AP*CP*TP*G)-3')B [auth T],
E [auth F]
11synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
DNA (5'-D(*CP*AP*GP*TP*AP*C)-3')C [auth P],
F [auth E]
6synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 4
MoleculeChains LengthOrganismImage
DNA (5'-D(P*GP*CP*CP*G)-3')G [auth D],
H [auth C]
4synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.234 
  • R-Value Work: 0.202 
  • R-Value Observed: 0.203 
  • Space Group: P 21 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 93.003α = 90
b = 148.712β = 90
c = 86.414γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-2000data collection
HKL-2000data scaling
PDB_EXTRACTdata extraction
HKL-2000data reduction
PHENIXphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2016-02-24
    Type: Initial release
  • Version 1.1: 2016-03-09
    Changes: Database references
  • Version 1.2: 2023-11-08
    Changes: Data collection, Database references, Derived calculations, Refinement description