Linear mitochondrial DNAs of yeasts: closed-loop structure of the termini and possible linear-circular conversion mechanisms.

Abstract

The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined. By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia piperi and P. jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA. The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism. Contiguous to the terminal loops, inverted terminal repeats were present. The mtDNA from WlUiopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated. Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length. Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed. A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.In the accompanying report (8), we show that linear forms of mitochondrial DNA (mtDNA) are frequently found in yeast species. These genomes have a defined terminal structure, as judged from their constant gene order with respect to the termini and the presence of homologous sequences at both termini. In this study, we analyzed in more detail the structure of the termini, as this information is essential to know the origin and mode of replication of the linear genomes. In a number of other organelle genomes, linear forms of DNA have also been identified. In some cases, the reality of the linearity might be questioned. In the case of Pichia pijperi, which had the smallest linear mtDNA, we could demonstrate, by sequencing, that the termini of the linear molecule possessed a continuous hairpin structure linking the termini of the two strands. The results obtained with two other species also suggested that an analogous structure may form the ends of their linear mtDNAs. MATERIALS AND METHODSStrains, media, and most of the analytical procedures are described in the accompanying report (8). Exonuclease III (ExoIII), nuclease Si, and nuclease BAL 31 were obtained from New England Biolabs (Beverly, Mass.) and used according to the supplier's instruction. The pTZ18/19R vector system (Pharmacia France, Saint Quentin-en-Yvelines, France) was used for cloning and enzymatic DNA sequencing. Electrophoresis of denatured DNA was carried out as described elsewhere (21). For electron microscopy, restriction fragments from termini were treated with the gene 32 protein of Eschenchia coli bacteriophage T4 as described previously (7) and then deposited on a carbon-coated grid that had been activated by glow discharge in the presence of pentylamine as described previously (7). mtDNA denaturation was performed according to the formamide-glyoxal method, and denatured DNA was spread as in the case of native DNA ...

Linear mitochondrial DNAs of yeasts: frequency of occurrence and general features.

“…Among 58 species examined (mostly type strains of the species), 16 species gave a distinct mtDNA band in PFGE, suggesting its linearity; all others gave no distinct band. Among the 16 species, 13 were confirmed to be linear by restriction mapping and later by enzymatic digestions of termini (see the accompanying report [4]). Three turned out to be circular, indicating that visualization by PFGE is not an absolute criterion of linearity, though highly indicative.…”

“…This was seen typically with P. pijpeni and P. jadinii mtDNAs. In W. mrakii, however, these faint bands are not visible because the shadow bands constitute the majority species of the terminal fragments (see the accompanying report [4]). The intensity of these shadow bands varied among the DNA preparations.…”

Rolling-Circle Replication of Mitochondrial DNA in the Higher Plant Chenopodium album (L.)

“…The generation of sigma-like molecules is usually taken as an indication of the occurrence of rolling-circle replication (11,12,15,27), although intermediates of the replication of linear mitochondrial genomes have a similar appearance in EM (13,17). Rolling-circle replication, via sigma-like intermediates, is the only process for which the generation of circular ssDNA molecules has been convincingly demonstrated (2,11,12,35).…”

“…However, the mtDNA of trypanosomatids is organized differently; it is present as giant catenanes of minicircles and maxicircles, with the individual molecules also replicating unidirectionally via theta-like intermediates (9). The replication of the linear mitochondrial genomes of Paramecium aurelia and certain fungi proceeds via lariat-shaped molecules (13,17). The mtDNA of S. cerevisiae is about five times larger than mammalian mtDNA.…”

Phage N15 Telomere Resolution

“…African swine fever virus (13)), and parvovirus (e.g. minute virus of mice (14)); and the genomes and certain plasmids of mitochondria and plastids (15)(16)(17). In none of these cases is replication clearly understood.…”