In vitro replication of heavy strand DNA in permeabilized human mitochondria

We have characterized some of the experimental conditions that are essential for initiation of human mitochondrial DNA synthesis. Mitochondria were purified from HeLa cells and were permeabilized with Triton X-100. When supplied with rNTPs and dNTPs, the permeabilized mitochondria synthesized nucleic acids that ranged in size from about 600 to 2000 nucleotides. In vitro DNA synthesis occurred on endogenous DNA templates and required a continuous supply of ATP. Analyses of the synthetic products revealed that almost all of them were of heavy-strand sequence and included authentic 7S DNA. Most of the synthetic products had 5' ends that mapped to similar locations as those previously identified for nascent heavy-strand DNA. Identification of these parameters should facilitate our efforts to achieve in vitro replication of heavy-strand mitochondrial DNA.