Mitochondrial diseases affect 1/2000 to 1/5000 people in the general population (1
). A wide spectrum of mitochondrial disease affects both children and adults. Mutations in nuclear genes encoding mitochondrial DNA (mtDNA) replication components, such as POLG
, have been associated with disease (2
). DNA polymerase γ (pol γ) is a heterotrimeric enzyme consisting of a 140 kDa catalytic subunit (encoded by POLG
) with high fidelity DNA polymerase and proofreading activities, and a homodimeric 55 kDa accessory subunit (encoded by POLG2
) for tight DNA binding and processive DNA synthesis (3
). Pol γ is the only DNA polymerase within the mitochondrion, thus it is essential for replication and repair of mtDNA. In particular, POLG
is a major locus for mitochondrial disease (see
), with more than 100 mutations associated with the fatal early childhood Alpers syndrome, midlife-onset ataxia neuropathy syndromes, late onset progressive external ophthalmoplegia (PEO), male infertility, and susceptibility to nucleoside reverse transcriptase inhibitor drugs commonly used to treat HIV (see http://tools.niehs.nih.gov/polg/
for references). Furthermore, mutations within the accessory subunit gene (POLG2
) are known to cause PEO (5
Known disease mutations in POLG.
We, and others have characterized a number of disease mutations, in order to determine the nature of the biochemical defects arising in these mutant proteins (6
). For example, the most common amino acid substitution in POLG
, A467T, was shown to have a severe catalytic defect, as both polymerase and exonuclease activities were greatly reduced compared to wild-type (WT) enzyme (11
). Interestingly, this mutation also disrupts physical association between the catalytic and accessory subunits (11
). As more mutant pol γ variants are analyzed the structural basis for the biochemical defects in each enzyme is becoming clear (12
). Likewise, the involvement of single nucleotide polymorphisms in disease has been found to be important. In the case of pol γ with both W748S and E1143G in the same allele, the E1143G single nucleotide polymorphism contributes both beneficial and detrimental effects to the protein (12
). Since more patients with mitochondrial disease are found with POLG
mutations every year, it is imperative that we understand the mechanisms by which these mutations cause mtDNA instability and ultimately mitochondrial disease, and the contributions of each mutation towards pathology.
By utilizing an extensive array of methods, we are developing a clearer understanding of how defects in pol γ contribute to disease. Furthermore, crucial knowledge concerning the role of pol γ in mtDNA replication and repair can be acquired. Here we present the protocols we use in the laboratory to characterize mutant DNA pol γ proteins, namely, assays to quantify polymerase activity, processivity, exonuclease activity, DNA binding, interaction between subunits, and protein stability.