Thymidine incorporation into deoxyribonucleic acid by isolated rat liver mitochondria.

Isolated rat liver mitochondria can incorporate tritiated thymidine into DNA. The addition of deoxyribonucleotides and ATP are necessary. The process of incorporation was found to be inhibited to the extent of 92% by actinomycin D and only 14% by pancreatic deoxyribonuclease. The identification of the labeled product as mitochondrial DNA is supported by its insolubility in acid, resistance to alkali, stability in the presence of RNase, hydrolysis when exposed to DNase, and buoyant density which was coincident with that of unlabeled mitochondrial DNA when determined in a density gradient of cesium chloride. Evidence that the 3H-thymidine was incorporated inside the DNA chain is provided by the parallel increase in acid-solubility of 3H and absorbance at 260 rnp when the acid-insoluble, labeled material was exposed to the action of DNase, phosphodiesterase, and 5’-nucleotidase. The specific activity of the resulting 3H-thymidine was similar at three times during the hydrolysis. Tritiated, phosphorylated derivatives of thymidine were isolated from the acid-soluble fraction of the mitochondria after incubation with 3H-thymidine. Thymidine is apparently converted to TTP via formation of TMP and probably TDP. It was estimated that only 0.4 to 0.5% of the total incorporation of 3H-thymidine could be accounted for by the level of bacterial contamination present in the mitochondrial preparation.