Functional analysis of mutant mitochondrial DNA polymerase proteins involved in human disease.

DNA polymerase gamma (pol gamma) 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 gamma, 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 gamma defects lead to disease. By using an extensive array of methods, we are developing a clearer understanding of how defects in pol gamma contribute to disease. Furthermore, crucial knowledge concerning the role of pol gamma in mtDNA replication and repair can be acquired. Here we present the protocols to characterize mutant DNA pol gamma proteins, namely, assays for processive DNA synthesis, exonuclease activity, DNA binding, subunit interaction, and protein stability.