Biochemical, cellular and molecular identification of DNA polymerase α in yeast mitochondria

Abstract

DNA replication occurs in various compartments of eukaryotic cells such as the nuclei, mitochondria and chloroplasts, the latter of which is used in plants and algae. Replication appears to be simpler in the mitochondria than in the nucleus where multiple DNA polymerases, which are key enzymes for DNA synthesis, have been characterized. In mammals, only one mitochondrial DNA polymerase (pol γ) has been described to date. However, in the mitochondria of the yeast Saccharomyces cerevisiae, we have found and characterized a second DNA polymerase. To identify this enzyme, several biochemical approaches such as proteinase K treatment of sucrose gradient purified mitochondria, analysis of mitoplasts, electron microscopy and the use of mitochondrial and cytoplasmic markers for immunoblotting demonstrated that this second DNA polymerase is neither a nuclear or cytoplasmic contaminant nor a proteolytic product of pol γ. An improved purification procedure and the use of mass spectrometry allowed us to identify this enzyme as DNA polymerase α. Moreover, tagging DNA polymerase α with a fluorescent probe demonstrated that this enzyme is localized both in the nucleus and in the organelles of intact yeast cells. The presence of two replicative DNA polymerases may shed new light on the mtDNA replication process in S. cerevisiae.