Functional Analysis of Mutant Mitochondrial DNA Polymerase Proteins Involved in Human Disease

DNA polymerase γ (pol γ) is the only DNA polymerase within the mitochondrion and is thus essential for replication and repair of mtDNA. POLG , the gene encoding the catalytic subunit of pol γ, is a major locus for a wide spectrum of mitochondrial diseases with more than 100 known disease mutations. Thus, we need to understand how and why pol γ defects lead to disease. By using 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 to characterize mutant DNA pol γ proteins, namely, assays for processive DNA synthesis, exonuclease activity, DNA binding, subunit interaction, and protein stability.