Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

Abstract

Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of the mitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization.

Roles of the mitochondrial replisome in mitochondrial DNA deletion formation

“…Another possibility considered by Sfeir and coworkers was an uncontrolled activity of DNA metabolic enzymes. In agreement with this idea, in our recent study on mtDNA replication in Drosophila melanogaster, we observed that an elevated expression of wild-type Twinkle resulted in the accumulation of DmtDNA, which expanded clonally to the extent that no wild-type mtDNA was detected by Southern-blotting (Ciesielski et al, 2018). Deletions have also been reported for mice overexpressing the wild-type helicase (Ylikallio et al, 2010).…”

“…Earlier reports did in fact indicate low levels of DSB repair in mitochondria (Bacman et al, 2009;Fukui and Moraes, 2009). Overall, the correlation between the frequency of DSBs and deletion formation in mitochondria appears to be well documented (Fukui and Moraes, 2009;Ciesielski et al, 2018;Nissanka et al, 2018), and in the light of these reports, including our own studies, they are very likely related. Perhaps, the coexistence of the rapid degradation of linearized mtDNA and the low efficiency of DSB repair could actually explain the clonal expansion phenomenon, as the rapid degradation of many cleaved molecules (which could arise in abundance, for example, from uncoupling of the mitochondrial replisome, as discussed in the next section) would give advantage to the replication of those few repaired DmtDNA, which consequently would repopulate mitochondria.…”

“…Markedly, the effects of overexpressing Twinkle in fly cells resemble the effects of halting Pol g in human cells with dideoxynucleotides, to which the mitochondrial replicase is highly sensitive. In both cases, RIs are consistent with replication stalling and exhibit long stretches of ssDNA, as well as substantial incorporation of RNA (Wanrooij et al, 2007;Ciesielski et al, 2018). These features are consistent with desynchronization between unwinding of the template DNA and DNA synthesis per se.…”

“…Homologous recombination is one of the fundamental DSB repair mechanisms in a wide spectrum of genetic sys- tems, which made the recombination-mediated mechanism of DmtDNA formation plausible, although historically lack of recombination has been erroneously associated with animal mtDNA (Harrison, 1989;Avise, 2000). The model was further supported by circumstantial evidence for recombination in mitochondria, including the concurrent accumulation of linear and DmtDNA forms, the observation of four-way junctions, catenates and other recombination intermediates, the identification of specific nuclear recombination factors in mitochondria, and others (Srivastava and Moraes, 2005;Bacman et al, 2009; de Souza-Pinto et al, 2009; Fukui and Pohjoismäki et al, 2009Pohjoismäki et al, , 2011Sage et al, 2010;Ciesielski et al, 2018). Perhaps the most compelling evidence comes from a study that demonstrated that mitochondrial protein extracts from distinct rat tissues and from HeLa cells were able to mediate joining of DNA substrates bearing microhomologies between 5 and 22-nt, which are similar to the flanking regions of the class I and II deletions (Tadi et al, 2016).…”

“…It has been suggested in several reports that both mechanisms, the replication-slippage and the DSB repair, may coexist and facilitate the formation of distinct deletion classes; e.g., replication-slippage would account for the class I deletions, whereas DSB repair would lead to classes II and III, or, alternatively, that replication-slippage may be specific to sporadic deletions, and DSB repair to the secondary deletions (Degoul et al, 1991;Mita et al, 1990;Wanrooij et al, 2004;Tuppen et al, 2010;Chen et al, 2011;Ciesielski et al, 2018). However, comparative studies of all three classes of deletions revealed remarkable similarities in the distribution of deletion breakpoints, which were independent of the presence of repeat sequences (Samuels et al, 2004;Reeve et al, 2008;Guo et al, 2010).…”