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

ABSTRACT

Mitochondrial DNA (mtDNA) of Saccharomyces cerevisiaecontains highly conserved sequences, called rep/ori, that are associated with several aspects of its metabolism. Theserep/ori sequences confer the transmission advantage exhibited by a class of deletion mutants called hypersuppressive petite mutants. In addition, because they share features with the mitochondrial leading-strand DNA replication origin of mammals, rep/ori sequences have also been proposed to participate in mtDNA replication initiation. Like the mammalian origins, where transcription is used as a priming mechanism for DNA synthesis, yeast rep/ori sequences contain an active promoter. Although transcription is required for maintenance of wild-type mtDNA in yeast, the role of the rep/ori promoter as a cis-acting element involved in the replication of wild-type mtDNA is unclear, since mitochondrial deletion mutants need neither transcription nor a rep/ori sequence to maintain their genome. Similarly, transcription from the rep/oripromoter does not seem to be necessary for biased inheritance of mtDNA. As a step to elucidate the function of the rep/oripromoter, we have attempted to detect transcription-dependent DNA transactions in the mtDNA of a hypersuppressive petite mutant. We have examined the mtDNA of the well-characterized petite mutant a-1/1R/Z1, whose repeat unit shelters the rep/ori sequenceori1, in strains carrying either wild-type or null alleles of the nuclear genes encoding the mitochondrial transcription apparatus. Complex DNA transactions were detected that take place around GC-cluster C, an evolutionarily conserved GC-rich sequence block immediately downstream from the rep/ori promoter. These transactions are strictly dependent upon mitochondrial transcription.

ACKNOWLEDGMENTS

We are grateful to H. P. Zassenhaus for the gift of plasmid pUZ5. We thank the members of the Clayton lab for stimulating discussions and Michel Ghislain, Denise Thinès, Françoise Foury, and André Goffeau (Louvain-La-Neuve) for their continuous support during the redaction of the manuscript.

E.V.D. was supported by Fogarty International Center Postdoctoral Fellowship 1FO5TWO4940. This work was supported by National Institute of General Medical Sciences grant R37-GM33088-27.

ADDENDUM IN PROOF

A very recent and precise mapping of RNA primers of yeastori5 petite mutation mtDNAs shows that the ori5promoter is the start site for replication (Nucleic Acids Res.26:1309–1316, 1998), consistent with the model for mammalian mtDNA replication.