A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans

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.

Inhibition of ATR Reverses a Mitochondrial Respiratory Insufficiency

“…Consistent with this idea, in mouse embryonic fibroblasts, mitochondrial stress results in mtDNA leaking into the cytoplasm and initiating an immune response 63 . On the other hand, worms use a rolling circle replication method for their mtDNA, so precisely how R-loops would form in the absence of D-loop (theta) replication, as is used by mammalian cells, remains enigmatic 64 . Another idea is that the low molecular weight species we detected are cytoplasmic DNA::RNA hybrids produced by DNA polymerase α (DNA pol α).…”

Animal Mitochondrial DNA Replication

“…The nematode mtDNA polymerase resembles the enzyme of other eukaryotic taxa such as Saccharomyces cerevisiae . Notably, mtDNA replication in the nematode C. elegans proceeds via a rolling circle mechanism [66], which is apparently the main mode of mtDNA replication in S. cerevisiae [67], and represents a mechanism distinct from that shown for other animals (see Section 3).…”

“…These are representative of fragments containing elongating replication forks and cruciform structures, respectively [79–82]. No structure corresponding to a replication initiation bubble (bubble arc) was detected [66]. Direct observation of the isolated mtDNA by transmission electron microscopy (TEM) identified branched-circular lariat molecules with concatemeric tails, which are characteristic of intermediates during rolling circle replication (Figure 7).…”

“…Interestingly, mitochondria of plants [91, 92] and fungi [67, 93] employ this mechanism of DNA replication, which suggests it’s ancestral origin. Nematodes represented by C. elegans appear to have maintained it [66], although all other animals analyzed to date seem to have evolved distinct mechanisms to replicate their mtDNA (see below).…”

“…A , The rolling circle mechanism of nematode mtDNA replication proposed by Lewis et al [66]. Circular progeny mtDNA molecules are proposed to be formed by a recombination-based resolution that involves the major non-coding region (NCR).…”