Amphimeric mitochondrial genomes of petite mutants of yeast. III. Generation by linking two secondary-structure-dependent illegitimate recombination events.

The generation of amphimeric mitochondrial petite genomes of yeast can be explained by a process that links together two illegitimate recombination events, each involving a pair of short inverted repeats. Following "diagonal" double-strand breaks and inter-strand ligations at both possible stem-and-loop structures, a subgenomic single-stranded DNA circle can be excised. This circle comprises four building blocks organized in the so-called datA arrangement where d and t correspond, respectively, to the segments looped out by the upstream and the downstream pair of inverted repeats, a to the sequence separating the two loops, and A to the inverted duplication of segment a. Depending on the different possible "diagonal" recombinations at the inverted repeats, any of four isomeric circles can be excised, representing in its double-stranded form the nascent basic unit of an amphimeric mitochondrial petite genome of yeast. These isomeric basic units differ in the relative orientation of their sequences d and t (called D and T, respectively, when inverted), and are designated datA, DatA, daTA, and DaTA. Any one of these may be replicated to form the previously described regularly arrayed multimeric flip-flop genomes. Our new understanding of the amphimeric mitochondrial petite genomes of yeast emphasizes the role that topological features of DNA can play in mitochondrial genome dynamics. It also permits the re-interpretation of various observations reported in the literature. Some of them, including EtBr-mutagenesis in yeast, are discussed.

Organization, generation and replication of amphimeric genomes: a review.

Genomes comprising a pair of separated inverted repeats and called 'amphimers' are reviewed. Amphimeric genomes are observed in a large variety of different organisms, ranging from archaebacteria to mammals. The widespread existence of amphimeric genomes in nature could be due to their particular dynamic structure. Amphimeric genomes containing long inverted segments may provide the only form in which a duplicated segment is stably retained in genomes. Amphimers are often found in amplified subgenomes, indicating that they could promote a special mechanism of DNA replication and amplification. The possible mechanisms of generation, isomerization and replication/amplification of different types of amphimeric genomes are discussed. The study of amphimeric mitochondrial petite genomes of yeast could be a good model system for the study of the role of inverted repeat sequences in genome dynamics.

Amphimeric mitochondrial genomes of petite mutants of yeast. I. Flip-flop amphimers make up the mitochondrial genomes of "palindromic" petite mutants of yeast.

The mitochondrial (mt) genomes of three spontaneous cytoplasmic "palindromic" petite mutants of yeast were studied by restriction-enzyme analysis. These mt genomes were shown to be made up of an amplified "master basic unit" consisting of two inverted segments (a and A) and of two different unique segments (d and t) separating them. The basic unit was called "amphimeric", this term having been first proposed for certain lambda-phage mutants. We propose that in the mt genomes of the petite mutants studied, the four possible variants of the amphimeric basic unit form two - "flip" and "flop" - tetra-amphimeric repeat units datA-datA-DaTA-DaTA and DatA-DatA-daTA-daTA, respectively. These repeat units make two types of "amphimeric" mt genomes which exist in equal proportions in the cell. In each mt genome, the duplicated segment regularly alternates in its direct and inverted orientation (a...A...a...A...), whereas the unique segments are arranged twice in tandem fashion and twice in inverted fashion (d...d...D...D...d...d...andt...t...T...T...t...t...). The only difference between flip and flop amphimeric mt petite genomes is the different relative orientation of the unique segments in the mono-amphimers. In the mono-amphimers of flip mt genomes, both unique segments are arranged in the same direction (d...t and D...T), whereas in the mono-amphimers of flop mt genomes, both unique segments are arranged in opposite directions (D...t and d...T). Control experiments on one spontaneous petite mutant (which was an ancestor of the mutants studied here) and on three independent, previously investigated, EtBr-induced mutants showed that all of them were, in fact, organized in the same way. Analysing our experimental data and the results published by others, we conclude that amphimeric organization is a general feature of mt petite genomes of yeast previously called "palindromic" or "rearranged".

Amphimeric mitochondrial genomes of petite mutants of yeast. II. A model for the amplification of amphimeric mitochondrial petite DNA.

A model for the recombination-directed replication and amplification of the mtDNA of amphimeric petite mutants of S. cerevisiae is proposed. Replication of an amphimeric master basic unit datA would be initiated in the inverted components a and A. The initiation of replication should be associated with the amphimeric structure of the master basic unit itself, but could be promoted by the presence of ori sequences or of sequences facilitating the initiation of replication in the inverted duplications. The amplification unit of amphimeric genomes is considered to be the double-stranded circular hetero-diamphimer datA-DaTA. Amplification of both diamphimeric strands involves an invasion of the 3' ends of the newly synthesized strands into symmetrical homologous duplex DNA regions promoting the continuation of replication, and leads to the accumulation of two ("flip" and "flop") types of multi-amphimers. We consider that this mode of amplification represents a modified rolling-circle mechanism. By analogy, we propose to call our model of amplification the "rocking-circle model". This model is likely to apply to other genomes organized as amphimeric structures.

Lack of site-specific recombination between mitochondrial genomes of petite mutants of yeast.

Previous work from our laboratory showed that mitochondrial (mt) genomes, with tandem repeat units, from spontaneous, cytoplasmic petite mutants of Saccharomyces cerevisiae do not exhibit site-specific recombination in petite x petite crosses [Rayko et al., Gene 63 (1988) 213-226]. Here, we have extended and confirmed these observations by studying other crosses of petites with tandem repeat units, as well as crosses in which one of the parents was, instead, an unstable petite, a-15/4/1, having a palindromic mt genome. In no case was site-specific recombination of the parental mt genomes observed. Progeny cells harbored mt genomes derived from either one or both of the two parents, as shown by analysis of restriction fragments. In the case of biparental inheritance, extensive subcloning of the diploids showed that this was due to a persistent heteroplasmic state and not to intermolecular recombination. The 'new' restriction fragments present in the mt DNA from some diploids were shown to be derived from the unstable parental genome, a-15/4/1, by a secondary excision process. Lack of site-specific recombination is, therefore, not only a feature of crosses involving petite genomes made up of tandem repeat units, but also of crosses in which one parental genome consists of inverted repeats and frequently originates secondary petite genomes formed by tandem repeats. Previous reports of mt recombination in petite mutants are discussed in light of these results.

Regions flanking ori sequences affect the replication efficiency of the mitochondrial genome of ori+ petite mutants from yeast.

The mitochondrial genomes of progenies from 26 crosses between 17 cytoplasmic, spontaneous, suppressive, ori+ petite mutants of Saccharomyces cerevisiae have been studied by electrophoresis of restriction fragments. Only parental genomes (or occasionally, genomes derived from them by secondary excisions) were found in the progenies of the almost 500 diploids investigated; no evidence for illegitimate, site-specific mitochondrial recombination was detected. One of the parental genomes was always found to be predominate over the other one, although to different extents in different crosses. This predominance appears to be due to a higher replication efficiency, which is correlated with a greater density of ori sequences on the mitochondrial genome (and with a shorter repeat unit size of the latter). Exceptions to the 'repeat-unit-size rule' were found, however, even when the parental mitochondrial genomes carried the same ori sequence. This indicates that noncoding, intergenic sequences outside ori sequences also play a role in modulating replication efficiency. Since in different petites such sequences differ in primary structure, size, and position relative to ori sequences, this modulation is likely to take place through an indirect effect on DNA and nucleoid structure.

Nuclear mutations affecting the stability of the mitochondrial genome in S. cerevisiae.

We have studied a pleiotropic mutation petD in S. cerevisiae which both confers the inability to grow on glycerol (Gly-) and greatly increases the frequency of cytoplasmic petites (Het). The first phenotype, Gly-, is recessive, whereas the second, Het, is dominant. Genetic and biochemical analysis showed that the majority of the petites in petD strains are not of the rho degree type (completely lacking mit-DNA), but of the rho- type (containing partially deleted mit-DNA). This finding and the fact that the phenotype Het is dominant argue in favour of the involvement of the petD product in the excision process of the mit-DNA. Another nuclear mutation, mod, was shown to exhibit a dominant epistasy with respect to the Het phenotype of the mutation petD. Two types of Gly+ revertants from petD mutants were isolated: rpa revertants, which restore completely the wild-type phenotype, and rpb revertants, which restore only the growth on glycerol, but still allow the production of high frequencies of cytoplasmic petites. Thus the mutations mod and rpb permit the genetic uncoupling of two phenotypes induced by the mutation petD.