Abstract:
The CCE1 (Cruciform Cutting Endonuclease) gene in S. cerevisiae
codes for an endonuclease that recognizes and cleaves recombination junctions
in mitochondrial DNA (mtDNA). In the absence of an active endonuclease, the
mitochondrial genomes remain interconnected and fail to segregate efficiently
into the daughter cells. Recently a homolog of CCE1 from S. pombe has been identified,
termed YDC2. To determine whether the YDC2 enzyme also plays a role in the maintenance
of mtDNA, we examined the effect of GAL1 induced overexpression of CCE1 and
YDC2 in S. cerevisiae cells. Transformants were grown for 6-8 generations in
either glucose (non-inducing) or galactose (inducing) medium. Loss of mtDNA
was determined by DAPI staining, and by a respiration-dependent colony color
assay. Under non-inducing conditions, the frequency of cells without mtDNA was
low: less than 3% of the culture. When grown in the presence of galactose, however,
the number of cells devoid of mtDNA for both YDC2 and CCE1 transformants was
much greater: over 30% of the culture. Vector-only controls did not show an
appreciable increase in mtDNA loss under inducing conditions. A mutant version
of YDC2 that binds to recombination junctions, but is unable to resolve them,
also produced a 10-fold increase in respiration deficient cells. These results
indicate that there is functional homology between the two resolvase enzymes.