Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Joachim M. Gerhold,Tiina Sedman,Katarina Visacka,Judita Slezakova,Lubomir Tomaska,Jozef Nosek,Juhan Sedman

doi:10.1074/jbc.m114.552828

Joachim M. Gerhold, Tiina Sedman + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m114.552828

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Abstract

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.

Highlights

Faithful mitochondrial DNA replication ensures functional oxidative phosphorylation
We demonstrate that mitochondrial DNA (mtDNA) intermediates in C. parapsilosis closely resemble those found in C. albicans despite the fact that the topological organization of the mtDNA is strikingly different
Oligonucleotides are listed that were used as oligos probe or as PCR primers on purified C. parapsilosis mtDNA to generate the respective PCR products to be used as double-stranded probes

Summary

Background

Faithful mitochondrial DNA replication ensures functional oxidative phosphorylation. Results: Recombination structures and replication forks are the main intermediates detected in Candida parapsilosis mtDNA. The presence of linear DNA molecules terminating in specific telomeric structures in the mitochondria of phylogenetically different yeast taxa evokes intriguing questions concerning their evolutionary origin [8, 36, 37]. To address these questions, we employed Candida parapsilosis that has a linear mtDNA terminating in arrays of telomeric repeats (30,923 ϩ 2n ϫ 738 bp, where n ϭ 0 –12) (Fig. 1A) [38, 39]. These hybrid molecules fall under the wider definition of RITOLS (RNA incorporation throughout the lagging strand) intermediates detected in mammalian mtDNA [41, 42] and demonstrate that stable RNA:DNA hybrids can be found in yeast mitochondria

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION