Double-forked circular molecules of mitochondrial DNA (mtDNA) from rat tissues, indicated by their form and size to be replicative intermediates, are of two structurally distinct classes. Molecules of the first class are totally double stranded. Molecules of the second class are defined by one daughter segment being totally or partially single stranded. Length histograms of daughter segments measuring between 2% and 44% of the total 5-µm molecular contour were constructed from samples of both classes of replicating molecules derived from mtDNA or Novikoff rat ascites hepatoma cells. For single strand-containing molecules, the lengths fell into eight distinct, reproducible groups with mean values separated by 4.1–7.6% of the circular contour length. For totally double stranded molecules, the lengths fell into seven groups, corresponding to seven of the groups found for single strand-containing molecules. These results suggest that along at least 44% of the contour of mtDNA molecules there exist discrete points at which DNA synthesis tends to be arrested. This may indicate that there are pauses in normal mtDNA synthesis. However, as the DNA used in these experiments was isolated from mitochondrial fractions, the findings may indicate that continuation of synthesis beyond specific points on the nucleotide strands requires a factor which is not available after cell disruption.

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