Abstract
Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.
Highlights
Caenorhabditis elegans is a ubiquitous model animal often employed in studies of aging and metabolic disease, processes intimately associated with mitochondrial health
Defects in the mitochondrial DNA that encodes protein subunits of the respiratory complexes may cause severe metabolic disease in humans. Such defects are often caused by errors during mtDNA synthesis, motivating ongoing studies of this process
We analyze the mechanism of mtDNA synthesis in the C. elegans gonad and demonstrate that it is unique among animals
Summary
Caenorhabditis elegans is a ubiquitous model animal often employed in studies of aging and metabolic disease, processes intimately associated with mitochondrial health. Comparatively little is known of mtDNA maintenance in this organism [1,2]. Studies of mitochondrial DNA (mtDNA) replication in mammalian cultured cells supported a unidirectional strand displacement or ‘asymmetric’ model, producing partially single-stranded-DNA (ssDNA) intermediates [3,4]. The previously described animal mtDNA replication models share two features. Initiation of replication relies on elongation from a transcript-primed displacement loop (D-loop). The previous work on mtDNA synthesis has focused primarily on mammalian species; mtDNA maintenance elsewhere in the animal lineage remains poorly understood
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