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The single-strand-specific nuclease S1 from Aspergillus oryzae rapidly converts superhelical mitochondrial DNA (African Green Monkey cells, Vero ATCC; CCL 81) into nicked circular DNA. These nicked mitochondrial DNA molecules contain two nicks, one in each strand. The phosphodiester backbones are cleaved during this reaction at or near sites that are alkali-labile.In a second slow reaction the circular mitochondrial DNA is converted into a linear duplex DNA. Permutation tests indicate that this linear DNA represents a, nonpermutated collection of DNA molecules. These results suggest that two of the alkali-labile sites in the phosphodiester backbones of the mitochondrial chromosome are closely spaced on opposite strands and at specific positions.Mitochondria of animal cells contain circular chromosomes which possess alkali-sensitive sites in the phosphodiester backbones of the DNA strands. There is increasing evidence that at least part of the alkali-sensitive sites can be explained by the presence of ribonucleotides in the mature mitochondrial DNA [l -61. Therefore, the mitochondrial chromosome appears to have a defined primary structure which is determined not only by the sequence of the deoxyribonucleotides but also by the number, sequence and position of the inserted ribonucleotides. So far, these features distinguish the mitochondrial DNA as a unique example of a circular chromosome. The only other example of a mature circular chromosome containing ribonucleotides is the closed circular plasmid DNA that accumulates in Escherichia coti if protein synthesis is inhibited by chloramphenicol Under appropriate conditions nuclease S1 can cut superhelical closed circular DNA into linear DNA of unit length as demonstrated for various viral . In this report we show that a unique collection of linear molecules is generated by the action of nuclease S1 on closed circular mitochondrial DNA. During this process the phospho-

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