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Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia

Nature Structural & Molecular Biology volume 11, pages 770776 (2004) | Download Citation

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Abstract

Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with mutations in the POLG gene encoding the mitochondrial DNA polymerase (pol γ). Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol γ. A homology model of the pol γ catalytic domain in complex with DNA was developed to investigate the effects of these mutations. Two mutations causing the most severe disease phenotype, Y955C and R943H, change residues that directly interact with the incoming dNTP. Polymerase mutants exhibit 0.03–30% wild-type polymerase activity and a 2- to 35-fold decrease in nucleotide selectivity in vitro. The reduced selectivity and catalytic efficiency of the autosomal dominant PEO mutants predict in vivo dysfunction, and the extent of biochemical defects correlates with the clinical severity of the disease.

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Acknowledgements

We thank S. Clark for technical assistance with production of the mutant derivatives and thank T. Kunkel and J. Santos for critical reading of this manuscript.

Author information

Affiliations

  1. Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

    • Maria A Graziewicz
    • , Matthew J Longley
    •  & William C Copeland
  2. Scientific Computing Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

    • Rachelle J Bienstock
  3. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland.

    • Maria A Graziewicz
  4. Unit of Molecular Neurogenetics, National Neurological Institute Carlo Besta, via Temolo 4, 20126 Milan, Italy.

    • Massimo Zeviani

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to William C Copeland.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    An example of a thermolysin digestion experiment of wild type DNA polymerase γ and one of its mutant derivatives, R943H.

  2. 2.

    Supplementary Fig. 2

    DNA-binding assay of the mutant pol γ polymerase.

  3. 3.

    Supplementary Table 1

    Single nucleotide mis-insertion assay with oligonucleotide substrates.

  4. 4.

    Supplementary Table 2

    Nucleotide mis-insertion assay with poly(dA)–oligo(dT)12–18.

  5. 5.

    Supplementary Table 3

    A·C mispair extension.

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DOI

https://doi.org/10.1038/nsmb805

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