The initiation of DNA replication in the mitochondrial genome of yeast

We report here the first direct demonstration that the active ori sequences of the mitochondrial genome of Saccharomyces cerevisiae are indeed origins of DNA replication, as previously postulated on the basis of compelling but indirect evidence. Basically, such sequences are formed by four regions: (i) GC clusters A and B, which are separated by a 29-bp AT stretch; (ii) a central 200-bp AT stretch, l; (iii) GC cluster C; (iv) a 16-bp AT stretch r, which comprises a site for transcription initiation. The ori sequences investigated, ori 1 and ori 5, have opposite orientations on the parental wild-type genome; ori 1 has but ori 5 does not have an additional 14-bp AT stretch r', between cluster C and sequence r; they were carried by the genomes of two spontaneous petites. In both ori sequences, nascent DNA chains using as template the strand containing sequence r (the 'r strand') start at the r end of cluster C, are elongated towards sequence l, and follow an RNA primer starting at sequence r. Nascent DNA chains copied on the 'non-r strand' start within cluster C, are elongated towards sequence r, and follow an RNA primer starting in sequence l just before cluster C. Ori 1 and 5 are, therefore, used as sites for RNA-primed bidirectional replication of mitochondrial DNA. Several aspects of this process are discussed.