Following synchronization by a double hydroxyurea block, mouse cell cultures exhibited a period of accelerated precursor incorporation into mitochondrial DNA during the late nuclear S phase. Peak activity of mitochondrial DNA polymerase-y occurred concurrent to the interval of accelerated organelle DNA synthesis. Mixing experiments suggested that the variations in mitochondrial DNA polymerase activity during the cell cycle were not due to free inhibitors in the enzyme preparations examined.There is well-documented evidence that mammalian cells possess in addition to the DNA polymerasesa, / 3 and y, a DNA polymerase activity associated with the mitochondria [l]. Recent reports on the characterization of this organelle DNA polymerase were, however, inconsistent with respect to the properties of the enzyme preparations described [2,3]. In contrast to earlier investigations by Kalf and Ch'ih [4] as well as Meyer and Simpson [5] and a recent report by Hecht [3] which deal with mitochondrial DNA polymerase from liver cells, the organelle enzyme from HeLa cells [2,6] did not exhibit a significant salt dependence and ethidium bromide sensitivity. Furthermore, a chloramphenicol treatment of HeLa cells was found to result in a considerable loss of this enzyme activity [7]. The latter observation could be interpreted in favour of the suspicion that the HeLa cell mitochondrial DNA polymerase is a procaryotic enzyme originating, for instance, from a mycoplasma contamination not detectable by routine examinations [7,8]. Attempts from this laboratory succeeded in isolating a DNA polymerase activity from a mitochondrial replication complex of mouse cells [8] which was most likely free of exogenous contaminations as a result of the isolation procedure. More than 98% of the activity solubilized from this complex showed distinct properties identical to those of the enzyme described by Hecht [3] purpose of this investigation was to look for a correlation between mitochondrial DNA synthesizing activity and the mitochondrial DNA polymerase activity during the cell cycle of synchronized mouse cells in order to further establish the function of the enzyme. It will be shown that there is a period of accelerated precursor incorporation into mitochondrial DNA [lo] during the cell cycle which is paralleled by the activity peak of the mitochondrial DNA polymerase-y. MATERIALS AND METHODS CellsCl-1D cells, a permanent mouse cell line [11,12] which is deficient in the major cellular thymidine kinase activity, although not deficient in the mitochondrial thymidine kinase, were used for all experiments. The monolayer cultures were cultivated in F-16 medium (Gibco) supplemented with 5 % calf serum, 200 units penicillin/ml and 10 pg 5-bromodeoxyuridine/ml. This cell line incorporates exogenous thymidine mainly into mitochondrial DNA and cannot replicate in medium containing hypoxanthine (1 5 pg/ ml), aminopterin (20 pg/ml) and thymidine (4 pg/ml). Routine examinations for mycoplasma contamination were found to be negative. Synchronization Pro...