In Vitro Analysis of mtDNA Replication.

Human mitochondrial DNA is a small circular double-stranded molecule that is essential for cellular energy production. A specialized protein machinery replicates the mitochondrial genome, with DNA polymerase γ carrying out synthesis of both strands. According to the prevailing mitochondrial DNA replication model, the two strands are replicated asynchronously, with the leading heavy-strand initiating first, followed by the lagging light-strand. By using purified recombinant forms of the replication proteins and synthetic DNA templates, it is possible to reconstitute mitochondrial DNA replication in vitro. Here we provide details on how to differentially reconstitute replication of the leading- and lagging-strands.