Unveiling the mystery of mitochondrial DNA replication in yeasts

Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing.This work was supported by the National Institute of Health (NIH) grants R01AG023731 and R21AG047400 to X.J.

The N-terminal intrinsically disordered domain of mgm101p is localized to the mitochondrial nucleoid.

The mitochondrial genome maintenance gene, MGM101, is essential for yeasts that depend on mitochondrial DNA replication. Previously, in Saccharomyces cerevisiae, it has been found that the carboxy-terminal two-thirds of Mgm101p has a functional core. Furthermore, there is a high level of amino acid sequence conservation in this region from widely diverse species. By contrast, the amino-terminal region, that is also essential for function, does not have recognizable conservation. Using a bioinformatic approach we find that the functional core from yeast and a corresponding region of Mgm101p from the coral Acropora millepora have an ordered structure, while the N-terminal domains of sequences from yeast and coral are predicted to be disordered. To examine whether ordered and disordered domains of Mgm101p have specific or general functions we made chimeric proteins from yeast and coral by swapping the two regions. We find, by an in vivo assay in S.cerevisiae, that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered domain of the coral protein cannot substitute for its yeast counterpart. Mgm101p is found in the mitochondrial nucleoid along with enzymes and proteins involved in mtDNA replication. By attaching green fluorescent protein to the N-terminal disordered domain of yeast Mgm101p we find that GFP is still directed to the mitochondrial nucleoid where full-length Mgm101p-GFP is targeted

Creating and curating an archive: Bury St Edmunds and its Anglo-Saxon past

This contribution explores the mechanisms by which the Benedictine foundation of Bury St Edmunds sought to legitimise and preserve their spurious pre-Conquest privileges and holdings throughout the Middle Ages. The archive is extraordinary in terms of the large number of surviving registers and cartularies which contain copies of Anglo-Saxon charters, many of which are wholly or partly in Old English. The essay charts the changing use to which these ancient documents were put in response to threats to the foundation's continued enjoyment of its liberties. The focus throughout the essay is to demonstrate how pragmatic considerations at every stage affects the development of the archive and the ways in which these linguistically challenging texts were presented, re-presented, and represented during the Abbey’s history

Domain organization for the <i>S.cerevisiae</i> and <i>A.millepora</i> Mgm101p sequences.

<p>The thick black line represents the predicted mitochondrial target signal. The green bar identifies the experimentally determined core region for <i>S</i>. <i>cerevisiae</i> and the corresponding region determined from the sequence alignment for <i>A. millepora.</i> The orange bars represent predicted disordered regions. Disorder predictions were carried our using three independent methods, IUPred, PONDR VSL2, and DISOPRED2. The sequences were aligned so that the beginning of the core region is in the same vertical position.</p

Nucleoids labelled with GFP.

<p><i>S. cerevisiae</i> M2915-7C transformed with (A) pCXJ8MGM101-GFP and (B) the deletion plasmid pCXJ8MGM101Δ99-269-GFP subcultured to GMM Ade,His,Leu for 24 h at 30°C before being photographed. The deletion leaves 98 amino acids at the N-terminus of Mgm101p consisting of a 22 amino acid mitochondrial targeting signal and 76 amino acids incorporating the ID region.</p

Complementation of the temperature sensitive mutant.

<p>A GlyYP plate with 1, M2915-7C <i>mgm101-1</i>ts, and M2915-7C transformed with pCXJ22 plasmids containing, 2, <i>S.cerevisiae MGM101</i>, 3, <i>A.millepora MGM101</i> with a <i>S.cerevisiae</i> mitochondrial targeting signal (A.m.ID-A.m.C)(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056465#pone-0056465-g003" target="_blank">Fig. 3</a>), 4, <i>S.cerevisiae</i> intrinsically disordered (ID) domain joined to <i>A.millepora</i> core region (S.c.ID-A.m.C) and 5, <i>A.millepora</i> ID region joined to <i>S.cerevisiae</i> core region (A.m.ID-S.c.C). The constructs all have a mitochondrial targeting signal sequence as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056465#pone.0056465.s002" target="_blank">Figure S2</a>. The plate was incubated at 35°C for 3 days before being photographed.</p

Distribution of phenotypes in segregants containing pCXJ22 <i>MGM101</i> plasmids.

*<p>1 tetrad contains 4 Gly⁻ spores.</p