Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

Abstract

The precise positions of the origin of replication ‡ ‡ Origin of mitochondrial DNA replication is defined in this paper as the position in the mitochondrial DNA sequence which corresponds to the 5′-end of the 7 S DNA. A considerable amount of evidence has indicated that replication of the heavy strand of mitochondrial DNA in animal cells starts by displacement synthesis of the initiation sequence (7 S DNA) and formation of the D-loop, and then continues by unidirectional expansion of the D-loop (Robberson & Clayton, 1972; Kasamatsu & Vinograd, 1973; Brown & Vinograd, 1974); however, there is no direct evidence that heavy-strand replication proceeds by extension of the 7 S DNA. and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.