Transcription-Dependent DNA Transactions in the Mitochondrial Genome of a Yeast Hypersuppressive Petite Mutant

The mitochondrial DNA (mtDNA) genome of Saccharomyces cerevisiae has been the subject of extensive genetic analysis over the years (reviewed in reference 16). Respiratory-deficient deletion mutants of mtDNA (called petite or [rho−] mutants) arise spontaneously at a high frequency. Only a small fragment of the ∼80-kb wild-type mtDNA genome ([rho+]) is retained in a petite genome, where it is amplified so that the mass of mtDNA in the petite genome is equivalent to that in a wild-type cell. The retained fragment is referred to as the repeat unit of the petite genome. Some petite mutants give rise to diploid progeny composed almost exclusively of petite mutants when crossed with cells harboring [rho+] mtDNA. These mutants, termed hypersuppressive (HS) petite mutants, are thought to present an mtDNA genome with a replicative and/or segregation advantage over [rho+] DNA. While the basis for this biased inheritance is still unknown, molecular characterization of HS genomes has provided important insights (3, 11, 12). All HS genomes consist of a tandem repetition of a short DNA fragment containing one of four highly conserved sequences that are all present in the [rho+] genome. These sequences, called rep or ori, comprise a 300-bp segment sharing about 80% identity. Embedded within this segment are three perfectly conserved GC-rich blocks (GC clusters A, B, and C), as well as a functional promoter for transcription, upstream from GC-cluster C. The integrity of GC clusters A and B is required for rep/ori activity; similarly, deletion of GC cluster C and its surrounding region inactivates the rep/ori sequence (25; reference 1 and references therein). The preferential transmission of HS genomes in crosses with [rho+] genomes has been postulated to reflect an advantage conferred by the increased density of rep/ori sequences in the amplified HS genomes. The rep/ori sequences have been posited as active origins of mtDNA replication, and hybridization studies have provided evidence that they function as bidirectional origins of replication (reference 2 and references therein). In addition, because their structural organization is reminiscent of that at the origin for leading-strand synthesis in the D-loop region of vertebrate mtDNA, yeast mtRNA polymerase has been proposed to play a role in producing primers for DNA synthesis at the rep/ori sequences (2; reviewed in references 7 and 38). In this light, GC cluster C, also found in the D-loop region of vertebrates (where it is called conserved sequence block II), promotes the polymerase-dependent formation of a persistent RNA-DNA hybrid upon transcription of origin-containing templates (44, 45). Formation of such a hybrid is thought to represent an important step in the sequence of events that lead to initiation of mtDNA synthesis (44, 45). A relatively simple machinery ensures mtDNA transcription in yeast cells (reviewed in references 26 and 40). The mtRNA polymerase is composed of a core catalytic subunit and a promoter recognition factor, sc-mtTFB. The nuclear genes encoding these components, RPO41 and MTF1, respectively, are essential for maintenance of [rho+] mtDNA, which is consistent with the role postulated for mtRNA polymerase in replication initiation. However, cells lacking a functional mitochondrial transcription apparatus are still able to propagate [rho−] genomes, and a rep/ori sequence need not be present in these deletion mutants (19–21). Thus, either alternative modes of replication exist that use other sequences and/or different mechanisms or transcription plays no role in [rho−] mtDNA replication. In principle this can be postulated for wild-type as well as [rho−] DNA, and therefore the priming of DNA replication in yeast mitochondria remains an open question. Transcription of HS genomes is initiated at the promoter present within rep/ori sequences in vivo (1), and the presence of an active promoter seems to be an important property of these genomes. In support of this view is the structure of three additional sequences closely related to the rep/ori sequences that are detected in wild-type mtDNA. However, these have never been recovered alone in screens for spontaneous HS petites (11). While these sequences also harbor GC clusters A, B, and C, their promoter has been inactivated by the insertion of a mobile GC-rich element called cluster gamma (11, 43). Why these sequences, which have been termed inactive origins, do not give rise to HS petite mutants is unknown, but at least two hypotheses can be put forth. First, activity of the promoter is required for the formation of an HS genome from a wild-type mtDNA (for instance, the excision and/or amplification of the repeat unit). Second, lack of a functional promoter causes a rep/ori sequence to lose its replication-segregation advantage so that, once formed, the deletion genome is rapidly competed by wild-type mtDNA molecules. The requirement of mtDNA transcription for preferential inheritance of HS mtDNA has recently been tested with cells carrying a deletion allele of the RPO41 gene (30). Since the mitochondrial transcription machinery is essential for maintenance of [rho+] mtDNA, such a requirement was measured in crosses between HS [rho−] and neutral [rho−] mutants (that is, petite mutants whose mtDNA does not harbor a rep/ori sequence and is not preferentially transmitted to the progeny of crosses with [rho+] cells). HS genomes were found to remain preferentially inherited despite the inactivation of RPO41, and it was therefore concluded that transcription from the rep/ori promoter is not necessary for the biased inheritance of HS genomes (30). However, the issue of whether inheritance of mtDNA obeys the same molecular rules in [rho−] × [rho−] and [rho+] × [rho−] crosses remains unsolved. Therefore, the possibility still exists that mtDNA transcription is required for preferential inheritance of HS genomes in crosses between HS petite mutants and wild-type cells. As a first step toward addressing the role played by mtRNA polymerase in the metabolism of rep/ori sequences, we have attempted to detect transcription-dependent DNA transactions in mtDNA of an HS petite mutant. In this study, we have investigated the mtDNA from the well-characterized petite mutant a-1/1R/Z1, whose 416-bp repeat unit harbors the rep/ori sequence ori1 (1, 2). We report the detection by direct labeling of free 5′ DNA ends of DNA strands that have the same polarity as transcripts derived from the promoter. The 5′ ends of these DNAs map downstream from the promoter, at the 5′ boundary of GC cluster C, a site where transitions from RNA to DNA have previously been proposed to occur (2). Gene disruption experiments were performed to demonstrate that production of these DNA strands is strictly dependent upon the presence of functional alleles of the RPO41 and MTF1 genes. In addition, restriction enzyme analyses and 3′-end-labeling experiments have revealed the presence of double-stranded breaks in the mtDNA of petite a-1/1R/Z1. These complex DNA transactions also depend upon the organelle transcription machinery, and their role in the metabolism of HS genomes will be discussed. We have tested the requirement of the DNA-binding protein ABF2, the yeast homolog of mammalian mitochondrial transcription factor A (mtTFA), which has been shown to be involved in wild-type mtDNA stability (14), for the maintenance of HS mtDNA. In contrast to what is observed with wild-type mtDNA under nonselective conditions (14), ABF2 is not required for the maintenance of HS mtDNA. Moreover, rpo41 abf2 double mutants are still able to propagate the ori1 genome of petite mutant a-1/1R/Z1, indicating that ABF2 is not required for mtDNA maintenance in the absence of mtRNA polymerase. Finally, the transcription-dependent mtDNA transactions reported in this study do not depend on ABF2, further indicating that unlike its mammalian homolog (reviewed in reference 40), the ABF2 protein plays a limited role in mtDNA transcription.