Replicating mitochondrial DNA from human, monkey and mouse cells. The superhelix density of the closed-circular...

Pulse-labeled replicating mitochondrial DNA molecules of three different cell lines, mouse NCTC 929 (ATCC;CCL 1), monkey Vero (ATCC;CCL 81) and human WISH (ATCC;CCL 25) were classified according to their sedimentation velocity in neutral sucrose gradients. Replicating molecules were isolated and and the newly synthesized strands were dissociated from the parental DNA strands. This procedure yielded parental closed circular duplex DNA with a higher sedimentation velocity in neutral sucrose gradients and higher absolute values of superhelix density in comparison with values for mature unreplicating supercoiled mitochondrial DNA. Replicating molecules with these properties, i.e. higher absolute superhelix densities of the parental strands, comprise 2--4% of the total mass of the mitochondrial DNA in all three cell lines. In addition, mouse cells contain a large proportion of D-loop mitochondrial DNA molecules (28 S). Upon dissociation of the 8-S single-stranded initiation sequence from the parental strands, these molecules yielded parental supercoiled duplex DNA with a superhelix density similar to that of mature supercoiled mitochondrial DNA. D-loop mitochondrial DNA represents about 30% of the total mass of the mitochondrial DNA in mouse cells. Therefore, two populations of replicating mitochondrial DNA can be distinguished in mouse cells. One of these accumulates as D-loop mitochondrial DNA with constant topological winding, while the other replicates in a manner similar to that of Vero and Wish cells under progressively decreasing topological winding of the parental strands.