Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae

Abstract

The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ− cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells.

Analysis of translating mitoribosome reveals functional characteristics of translation in mitochondria of fungi

“…The rRNA element H59 was deleted, and H53 is unstructured and displaced, which is complemented by acquisition of the mitochondria-specific protein mL67/Mhr1. This mitoribosomal protein is also known as mitochondrial recombinase 25 that mediates mtDNA replication 26 , repair 27 , and was detected in the mitoribosomal assembly survey 28 . It has a flexible extension of 25 residues, which is not present in S. cerevisiae, adjacent to the exit site and pointing toward the membrane plane ( Supplementary Figs.…”

Mitochondrial genetics revisited

“…RDR is also supported in S. cerevisiae by the facts that mutants in CCE1 (encoding the cruciform cutting endonuclease) reduce hypersuppressiveness (Lockshon et al, ) and affect mitochondrial inheritance (Piskur, ) and that double mutants in MHR1 and in CCE1 are unable to maintain mtDNA. But RDR may not be the only mechanism of mitochondrial genome replication in S. cerevisiae because double‐strand break initiation (Prasai, Robinson, Scott, Tatchell, & Harrison, ) and RNA‐primed initiation (Sanchez‐Sandoval et al, ) have also been reported at rep / ori sequences.…”

Discordant evolution of mitochondrial and nuclear yeast genomes at population level

“…However, the two parental organelles are kept physically separated and can only be in contact and recombine on a limited interaction surface [35]. Nevertheless, recombination is considered to be a critical mechanism for mitochondrial inheritance [36] with profound effect on patterns of mitochondrial genetic variation [21,37,38].…”