• Significance of the Nuclear Gene RAD54 in Mitochondrial Genome Stability of Saccharomyces cerevisiae

      Sia, Rey; Stoj, Melissa; The College at Brockport (2017-05-04)
      Mitochondria are essential organelles in eukaryotic organisms that synthesize the energy-providing molecule, ATP, through the process of oxidative phosphorylation. As explained by the endosymbiotic theory, mitochondria contain mitochondrial DNA (mtDNA), distinct from nuclear DNA (nDNA). When mitochondrial function is impaired, and mtDNA stability is compromised, detrimental neuromuscular and neurodegenerative disorders such as Mitochondrial Encephalomyopathy, Lactic acidosis and Stroke-like episodes (MELAS) and Leber’s Hereditary Optic Neuropathy (LHON) have the potential to occur. The purpose of this study was to determine the role of the nuclear gene RAD54 in maintaining mtDNA stability in the budding yeast, Saccharomyces cerevisiae. Although the role of Rad54p in maintaining nDNA stability is understood, its impact on mtDNA stability is relatively unknown. RAD54 is a member of the RAD52 epistasis group, coding for a protein vital to the initial steps of homologous recombination and double-stranded break (DSB) repair. Given that members of the RAD52 epistasis group have been shown to contribute to homologous recombination and DSB repair in mtDNA of S. cerevisiae, we hypothesized that loss-of-function RAD54 would decrease the rate at which homologous recombination in mtDNA occurred (Stein, Kalifa & Sia, 2015). A phenotypic respiration loss assay was performed in a rad54? strain to determine the frequency of spontaneous mutations in mtDNA that blocked the oxidative phosphorylation process. The mutant strain demonstrated a 1.56-fold decrease in spontaneous respiration loss when compared to wild type (p-value = 0.0574). Interestingly, previous research has demonstrated that the nature of these spontaneous mutations is due to large deletions in the mtDNA. To investigate the role of Rad54p in preventing these deletions from occurring, a direct repeat-mediated deletion (DRMD) assay was performed. The DRMD assay demonstrated a significant 3.23-fold increase in nDNA homologous recombination events (p-value = 0.0158) and a statistically insignificant 1.08-fold increase in mtDNA homologous recombination events (p-value = 0.8741) between rad54? and wild type strains. Given the present findings of this study, it appears the nuclear gene RAD54 does not play a significant role in maintaining mtDNA stability in respiration loss or DRMD assays.