The overall mode of mitochondrial DNA (mtDNA) replication is best understood for the mouse and human systems, and a general model of vertebrate mtDNA replication has been established (9). Mammalian mtDNA replicates by unidirectional synthesis from two distinct origins, the origin of heavy-strand replication and the origin of light-strand replication, which are located two-thirds of the genomic distance apart on the closed circle. The structural features and mechanisms of RNA priming are very different at these two origins (3,7,9,29,30). A characteristic hallmark of the origin of heavy-strand replication is the presence of three evolutionarily conserved sequence blocks (CSBs I, II, and III) at or near the 5' ends of newly synthesized heavy strands. It has been demonstrated for nearly all 5' ends of nascent heavy-strand DNAs that there are RNA species whose 3' ends map immediately adjacent to the DNA 5' ends. It has also been shown that 5' ends of these RNAs map at the initiation site of transcription from the major lightstrand promoter (3, 7). On the basis of these and other findings, Chang and coworkers hypothesized that transcripts from this promoter play a role in heavy-strand replication by serving as primers for DNA synthesis and that individual primer termini were generated by processing of a primary transcript (3, 7).Mammalian complementary to the origin of leading heavy-strand mtDNA replication (4, 6, 28). The endonuclease activity is present in both nuclear and mitochondrial fractions (2,5,14,17,28) and has been proposed to participate in mitochondrial primer RNA metabolism in vivo. The standard mitochondrial RNA (mtRNA) substrate for mouse and human RNase MRPs contains three short regions of sequence that are highly conserved in vertebrate species (CSBs I, II, and III). Initial characterization of the mouse RNase MRP activity demonstrated that the site-specific cleavage of mtRNA substrate in vitro occurred immediately adjacent to CSB II, and it was postulated that CSB II played a role in cleavage site specificity (4). Subsequent deletional analysis and saturation mutagenesis have determined basic substrate requirements for cleavage by mouse RNase MRP; CSB II and CSB III are essential for both efficient and accurate cleavage, whereas CSB I is not (2).In contrast to the mammalian system, our knowledge of yeast wild-type mtDNA replication initiation, at the molecular level, is less advanced. 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). It has also been proposed that the active on sequences of yeast mtDNA are ...