Abstract
The human mitochondrial DNA polymerase gamma (Pol-γ) is nuclearly encoded and is responsible for the replication and repair of the mitochondrial genome. Mutations S305R and P1073L in the POLG gene have been reported to be associated with early childhood Alpers syndrome. One patient harboring both mutations as compound heterozygous died at 2 years of age after disease onset at 9 months. Quantitative kinetic analysis on purified enzyme showed that the S305R mutation reduces the DNA binding affinity by 10-fold, and reduces the specificity constant (kcat/Km) for correct nucleotide incorporation by fourfold. It also causes a ∼threefold reduction in the excision rate to remove mismatched nucleotides. Compared to the wild-type Pol-γ, the S305R mutant showed no product formation in a reconstituted rolling circle replisome assay. Interestingly, the P1073L mutant exhibited wild-type activity in single turnover kinetics to quantify changes in kcat/Km, kcat, kexo, or processivity, and showed a twofold decrease in the net polymerization rate in the reconstituted replisome assay, while in yeast, P1073L caused a 60–70% mtDNA reduction in haploid cells. The heterozygous diploid yeast cells carrying S305R and P1073L mutations in trans showed ∼75% reduction of mtDNA content, relative to homozygous diploid cells with two wild-type alleles. Taken together, we show clearly in both the rolling circle and the humanized yeast system that the P1073L mutation caused significant defects in mtDNA replication, and our results suggest a role for P1073 in the functioning of the Pol-γ with the mitochondrial DNA helicase, and provide a rationale for understanding the physiological consequences of the S305R/P1073L compound heterozygote in humans.
Highlights
To date more than 200 mutations have been reported in the POLG gene and they are correlated with a variety of mitochondrial disorders, including Alpers syndrome, progressive external ophthalmoplegia (PEO), Parkinsonism, and other encephalomyopathies associated with mtDNA mutations, deletions, and depletions (Stumpf and Copeland, 2011)
We showed that S305R and P1073L mutations cause mtDNA replication defects by different kinetic mechanisms, and our findings provided a rationale to understand the physiological consequences of the S305R/P1073L compound heterozygote
With a given mutation one would like to define the changes in kinetics of polymerization and to understand how biochemical defects lead to the observed phenotype
Summary
To date more than 200 mutations have been reported in the POLG gene (see http://tools.niehs.nih.gov/polg/) and they are correlated with a variety of mitochondrial disorders, including Alpers syndrome, progressive external ophthalmoplegia (PEO), Parkinsonism, and other encephalomyopathies associated with mtDNA mutations, deletions, and depletions (Stumpf and Copeland, 2011). The progressive, late-onset neurodegenerative diseases (PEO, Parkinsonism, etc.) are often caused by mutations in the active site of Pol-γA (Stumpf and Copeland, 2011), which can lead to reductions in kcat/Km, kcat, and fidelity of replication (Batabyal et al, 2010; Estep and Johnson, 2011; Ziehr et al, unpublished). Manifestation of Alpers syndrome typically requires the presence of at least two recessive mutations in Pol-γ, usually in compound heterozygous states, and is mostly associated with mtDNA depletions (Wong et al, 2008). The effects of individual recessive mutations on protein function and detailed molecular mechanism of pathogenesis remain to be investigated
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