Mechanism of mitochondrial DNA replication in mouse L cells: Localization of alkali-sensitive sites at the two origins of replication

Under alkaline conditions which completely degrade RNA but leave DNA intact, only a few percent of the mitochondrial DNA molecules of mouse L cells remain as intact closed circles. Approximately one-third of the closed circular molecules are nicked only once or twice, and the remainder are nicked at several sites, producing a heterogeneous distribution of fragment lengths. We have compared the products of alkali treatment of replicative intermediates with those of nonreplicating molecules, and no variation in the pattern of alkali-sensitive sites was detected. The two strands of the mitochondrial DNA duplex are both sensitive to high pH. Alkaline treatment of the two largest BamHI restriction endonuclease fragments produces specific degradation products consistent with the presence of alkali-sensitive sites at both the heavy- and light-strand replication origins. These sites may represent residues of ribonucleotide priming of the asynchronously replicated strands of mouse mitochondrial DNA.