Mitochondrial DNA integrity is not dependent on DNA polymerase-β activity

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-β (Polβ), which exhibits both 5′-deoxyribose-5-phosphate (5′-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-γ (Polγ) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polβ or a “Polβ-like” enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polβ is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polγ and the other to Polβ, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polβ from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polβ activity. In a functional test for the presence of Polβ function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polβ null cells. In contrast, mtDNA from cells harboring mutations in the Polγ exonuclease domain that abolish proofreading displayed a ≥17-fold increase in mutation frequency. We conclude that Polβ is not an essential component of the machinery that maintains mtDNA integrity.