In vivo rearrangement of mitochondrial DNA in Saccharomyces cerevisiae.

Abstract

A revertant (SPR1) from a high-frequency petite strain of Saccharomyces cerevisiae has been shown by mapping and sequence analysis to have a rearranged mitochondrial genome. In vivo rearrangement has occurred through a subgenomic-recombination pathway involving the initial formation of subgenomic molecules in nascent petite mutants, recombination between these molecules to form an intermediate with direct repeats, and subsequent excision of the resident or symposed duplication to yield a molecule with three novel junctions and a changed gene order. Sequencing of the novel junctions shows that intramolecular recombination in each case occurs by means of G+C-rich short direct repeats of 40-51 base pairs. Mapping and sequence analysis also reveal that the SPR1 mitochondrial genome lacks three sectors of the wild-type molecule of 4.4, 1.7, and 0.5 kilobases. Each of these sectors occurs in nontemplate, base-biased DNA, that is over 90% A+T. Absence of these sectors together with a rearranged gene order does not appear to affect the phenotype of SPR1, as colony morphology and growth rate on a number of different substrates are not detectably different from the wild type. Lack of phenotypic change suggests that mitochondrial gene expression has not been noticeably disrupted in SPR1 despite deletion of the consensus nonomer promoter upstream from the glutamic acid tRNA gene. Dispensability of DNA sectors and the presence of recombinogenic short, direct repeats are mandatory features of the subgenomic-recombination pathway for creating rearrangements in baker's yeast mtDNA. It is proposed that, in other organisms, organelle genomes containing these elements may undergo rearrangement by the same steps.Mapping of mitochondrial genomes in yeasts has revealed that both rearrangements and length mutations are widespread (1). Indeed, such changes are prevalent in mitochondrial genomes of lower eukaryotes (2) As stated above, a special facet of the subgenomicrecombination pathway is that the original wild-type molecule must contain at least one segment that is dispensable. Potentially dispensable sectors of yeast mtDNA are the base-biased regions that comprise over half the 80-kb molecule (2, 7). These regions, up to 7 kb long in one instance, are greater than 90% A+T. Interspersed in these stretches of A+T are various G+C "clusters" of 20-50 base pairs (bp) (8). As described below, three dispensable base-biased sectors and six G+C clusters are involved in the creation of a rearranged mitochondrial genome.Formation of a mitochondrial genome containing direct duplications has been inferred from studies on high-frequency pelite-(hfp)-forming strains of Saccharomyces cerevisiae (9, 10). These respiratory competent strains have been obtained by mating nascent spontaneous petites (11). Characterization of mtDNA in hfp strains has suggested that the mitochondrial genome is composed of two subgenomic molecules (originating in the nascent petites) in equilibrium with a cointegrate (10). However, due to the large number of pet...

Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.

“…(9). This result contrasts with those of another study in which it was found that GC clusters of the al category are involved in the generation of different deletions (3). However, both sets of findings confirm the recombinogenic nature of GC clusters and indicate that mitochondrial recombinases involved in deletion exhibit some flexibility with regard to sequence specificity.…”

“…Recent developments in manipulating the mitochondrial genome have enabled us to generate strains deleted for large segments of base-biased sequences (3,4). This has allowed us to examine whether some of these regions influence mitochondrial biogenesis.…”

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

“…That the DSBs detected in this study occur around GC cluster C may not be surprising, since GC clusters of several sequence types in yeast mtDNA have been found to be recombinogenic (6,13,49). Further work is required to determine whether the transcription-dependent DSBs we have observed are correlated with recombination structures.…”

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

“…Subgenomic recombinations also take place at short and direct sequence repeats, a pattern consistent with the involvement of a recombination-excision mechanism. These events underlie the frequent generation of respiration-deficient petite mutants in S. cerevisiae, which stably transmits the extensively deleted Ϫ genomes (41)(42)(43). Similar repeat-mediated recombination events were seen in Neurospora crassa, which are manifested by the generation of plasmid-like supercoiled subgenomic circles in mitochondria (44).…”