Hypersuppressiveness, as observed inEukaryotic cells gain most of the energy required for cellular functions by oxidative respiration in mitochondria. These organelles contain hundreds to thousands of copies of a mitochondrial DNA (mtDNA) genome that encodes components essential for respiration and protein synthesis. Generally, mitochondrial alleles segregate during vegetative cell growth (vegetative segregation), and all copies of mtDNA within a cell or an individual generally have the same sequence (homoplasmy). In patients with mitochondrial myopathies, the progressive accumulation of mtDNA with large deletions leads to a heteroplasmic state in specific tissues (20; for review, see references 43 and 54). This phenomenon may be caused by the selective replication and/or segregation of mtDNA bearing the deletion; however, genetic analysis of mtDNA processes in mammals has been hampered by the strict maternal inheritance of mitochondria. The yeast Saccharomyces cerevisiae is a suitable model system because of its biparental mtDNA inheritance (14).The extremely biased inheritance of mtDNA with a large deletion is known in S. cerevisiae as "hypersuppressiveness. Two mechanisms for the initiation of mtDNA replication in yeast have been proposed: mitochondrial transcriptional RNA polymerase (Rpo41)-primed initiation and recombination-mediated initiation. Rpo41-primed DNA replication is similar to that observed in mammalian mitochondria. 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). However, this transcription-dependent process is not the sole mechanism for the initiation of mtDNA replication, since both the replication origin and RPO41 are dispensable for the maintenance of [rho Ϫ ] mtDNA (18,53). In addition, it has been reported that the selective inheritance of hypersuppressive [rho Ϫ ] mtDNA relative to nonhypersuppres-* Corresponding author. Mailing address: