Isolation from wheat mitochondria of a membrane-associated high molecular weight complex involved in DNA synthesis.

A high molecular weight mitochondrial DNA (mtDNA) replication complex, associated with the mitochondrial membrane, was isolated by sucrose gradient centrifugation from purified wheat embryo mitochondria. This complex comprised the mtDNA as well as enzyme activities involved in the replication and transcription of the organelle genome, such as DNA polymerase, RNA polymerase and topoisomerase type I. The isolated complex is active in mtDNA and mtRNA synthesis in vitro. Electron microscopy and lipid analysis confirmed the membrane origin of this complex. Enzyme activities are resistant to physiological ionic strengths, 0.1-0.2 M KC1, while the membrane-mtDNA association is resistant up to 1 M KC1. DNase treatment of the complex released the DNA polymerase activity while protease treatment solubilized mtDNA, suggesting the direct interaction of mtDNA with membrane protein(s). The use of a novel approach to detect mtDNA fragments specifically retained by the mitochondrial membranes after Sal I digestion of the complex suggests that specific mtDNA sequences anchor mtDNA to mitochondrial membranes.