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.
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Supplementary information
Supplementary Fig. 1
An example of a thermolysin digestion experiment of wild type DNA polymerase γ and one of its mutant derivatives, R943H. (PDF 274 kb)
Supplementary Fig. 2
DNA-binding assay of the mutant pol γ polymerase. (PDF 137 kb)
Supplementary Table 1
Single nucleotide mis-insertion assay with oligonucleotide substrates. (PDF 22 kb)
Supplementary Table 2
Nucleotide mis-insertion assay with poly(dA)–oligo(dT)12–18. (PDF 24 kb)
Supplementary Table 3
A·C mispair extension. (PDF 23 kb)
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Graziewicz, M., Longley, M., Bienstock, R. et al. Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia. Nat Struct Mol Biol 11, 770–776 (2004). https://doi.org/10.1038/nsmb805
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DOI: https://doi.org/10.1038/nsmb805
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