The initiation of DNA replication in the mitochondrial genome of yeast.

Abstract

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.

Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming.

“…DISCUSSION A subset of yeast mitochondrial promoters is located immediately adjacent to putative origins of replication; as such, they have been implicated in priming of DNA replication. A common feature of these replication origins is the presence of a short guanosine-rich region (GC cluster C/CSB II-like element) in the priming strand downstream of the transcription start site (1,10,13,26). This situation is similar to that for the vertebrate mtDNA leading-strand origin and suggests possible functional relationships between the yeast on/rep and vertebrate origin systems.…”

“…6A). These nucleotides are exactly at the mapped position of linkage between RNA and DNA in nucleic acid isolated from mitochondria of a hypersuppressive yeast strain containing a mtDNA ori5 sequence (1 …”

“…However, studies with hypersuppressive petite strains indicate that putative yeast mitochondrial replication origins (on [or rep {reference 12 and references therein}] sequences) are characterized by a 300-bp A+T-rich segment containing the following regions: a 16-bp A+T-rich sequence containing an active promoter for transcription initiation (termed r), a 17-bp GC cluster C located immediately downstream of the promoter, a central 200-bp A+T-rich stretch (termed C), and GC clusters A and B, which are separated by an A+T-rich region (1,10,13 (1,10,26). These include the existence of a functional promoter at or near the site of transition from RNA to DNA synthesis; the presence of a short, conserved G-rich sequence block at the origin (GC cluster C/CSB II [10,13]); and the occurrence of an additional conserved element, CSB I, in this region (10,13).…”

DNA Recombination-Initiation Plays a Role in the Extremely Biased Inheritance of Yeast [rho−] Mitochondrial DNA That Contains the Replication Origin ori5

“…In support of this mechanism, each mtDNA replication origin shares sequence similarity with the heavy-strand replication origin of mammalian mtDNA, including the presence of a transcription promoter and three GC-rich clusters (2, 13). RNA synthesized by Rpo41 from mtDNA replication-origin promoters has been detected, and an endoribonuclease that cleaves the synthesized RNA at sites that correspond to regions of transition from RNA to DNA synthesis has been detected (3,49,53). The intact replication-origin promoter is required for hypersuppressiveness (36).…”

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

“…This led to the assumption that yeast mtDNA replication initiation is an RNA-primed process (13,14). Although RNA-primed DNA strands have been detected in rho Ϫ mtDNA (12,15,16) (18). In addition, ori/rep elements do not represent an evolutionary conserved feature required for mtDNA replication (19,20) and they may originate from a mobile or selfish element that invaded and was amplified in the mitochondria of the genus Saccharomyces (21).…”