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Digestion of mouse L cell mitochondrial DNA with EcoRI restriction endonuclease produces two linear duplex fragments comprising 86.3 +/- 2.0% and 14.2 +/- 1.0% of the circular genome length (16,000 +/- 470 nucleotide pairs). Digestion of human HeLa cell mitochondrial DNA with EcoRI produces three linear duplex fragments comprising 49.2 +/- 1.0%, 44.4 +/- 0.9%, and 6.4 +/- 0.4% of the circular genome length (16,590 +/- 710 nucleotide pairs). These fragments are shown to be generated by cleavage in unique regions of the mouse and human mitochondrial DNAs. An electron microscopic analysis of partially replicated molecules cleaved by EcoRI establishes a unidirectional mode of DNA replication for L cell mitochondrial DNA. The origin for DNA replication is located on the larger EcoRI fragment at a position that is 1,890 +/- 250 nucleotide pairs (11.8 +/- 1.2% of the circular genome length) from the proximal restriction site. Replication proceeds unidirectionally away from this restriction site throughout the length of the larger EcoRI fragment. Analysis of L cell, D-loop mitochondrial DNA cleaved by EcoRI indicates that a unique sequence is synthesized in formation of the D-loop in these nonreplicating molecules. The origin of D-loop synthesis is located on the larger EcoRI fragment at a position 1,760 +/- 180 nucleotide pairs (11.0 +/- 1.1% of the circular genome length) from the proximal restriction site and is, therefore, the origin for unidirectional displacement replication.

Cleavage of Replicating Forms of Mitochondrial DNA by Eco RI Endonuclease