pyrophosphate and Parkinson-Disease

The human mitochondrial DNA polymerase (pol γ) is nuclearly encoded and is solely responsible for the replication and repair of the mitochondrial genome. The progressive accumulation of mutations within the mitochondrial genome is thought to be related to aging, and mutations in the pol γ gene are responsible for numerous heritable disorders including progressive external opthalmoplegia, Alpers syndrome, and parkinsonism. Here we investigate the kinetic effect of H932Y, a mutation associated with opthalmoplegia. Mutations H932Y and H932A reduce the specificity constant governing correct nucleotide incorporation 150- and 70-fold, respectively, without significantly affecting fidelity of incorporation or the maximum rate of incorporation. However, this leads to only a 2-fold reduction in rate of incorporation at a physiological nucleotide concentration (∼100 μm). Surprisingly, incorporation of T:T or C:T mismatches catalyzed by either H932Y or H932A mutants was followed by slow pyrophosphate release (or fast pyrophosphate rebinding). Also, H932Y readily catalyzed incorporation of multiple mismatches, which may have a profound physiological impact over time. His-932 is thought to contact the β-phosphate of the incoming nucleotide, so it is perhaps surprising that H932Y appears to slow rather than accelerate pyrophosphate release.

Topics: Base Pair Mismatch; Catalysis; Cloning, Molecular; Diphosphates; DNA; DNA-Directed DNA Polymerase; Genome, Mitochondrial; Histidine; Humans; Kinetics; Mitochondria; Mutagenesis; Mutation; Nucleotides; Parkinson Disease

Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis.

Topics: Alleles; Automation; Biotin; Case-Control Studies; Diphosphates; DNA Mutational Analysis; DNA Primers; Gene Frequency; Genetic Techniques; Genotype; Humans; Molecular Biology; Parkinson Disease; Reproducibility of Results; Sequence Analysis, DNA