• The Effects of RAD1 and RAD10 on Mitochondrial Stability in the Saccharomyces cerevisiae

      Sia, Rey; Pelletier, Michel; Hing, Huey; Seger, Christina; State University of New York College at Brockport (2016-07-21)
      The mitochondria have long been known as the powerhouse of the cell due to their essential role in oxidative phosphorylation to create energy. Mitochondria have their own genome separate from the nucleus, and may undergo mutations that lead to neuromuscular disease as well as account for some effects of aging. The effects and repair mechanisms of mutations to nuclear DNA have long been studied in order to map out the specific proteins and pathways involved. Nucleotide excision repair is a pathway involving a single-strand break which allows the template strand to be copied after removing the damaged bases. Rad1p and Rad10p are subunits of the nucleotide excision repair factor 1 (NEF1) which cleaves 5' of the site of damage. Single strand annealing is a repair pathway in which a double-strand break is detected and regions of homology recombine creating 3' flaps of nonhomology. Rad1p and Rad10p together form a flap endonuclease that specifically cuts at the 5' incision during single-strand annealing (Bardwell et al. 1994). The goal of this research focuses on determining if the nuclear genes RAD1 and RAD10 play a role in mitochondrial stability. The results of an assay measuring spontaneous respiration loss showed a 1.5 fold increase in rad10? strains from wildtype strains and a 1.2 fold increase in rad1? strains. The increase in respiration loss of rad10? strains was significant with a p value of 0.00003 in a two-tailed t-test. The increase in rad1? strain respiration loss was insignificant with a p value of 0.068 in a two-tailed t test. A direct repeat-mediated deletion (DRMD) assay was performed and resulted in a 2.3 fold decrease in nuclear mutation rate in rad10? strains compared to wildtype and a 1.8 fold decrease in rad1? strains. A two-tailed t-test was performed and indicated that the 2.3 fold decrease in nuclear mutation rates was significant with a p value of 0.0003 for rad10? strains. The 1.8 fold decrease in rad1? strain mutations was significant with a p value of 0.0005 in a two-tailed t test. The DRMD assay also indicated that there was no significant change in mutation rates in mitochondrial DNA of rad10?, or rad1? strains compared to wildtype. A two-tailed t-test demonstrated the insignificance of the mitochondrial mutation rate changes, a p value of 0.44 was obtained for rad1? and a p value of 0.73 was obtained for rad10?. Results from an induced DRMD assay showed a 1.25 fold increase in mutation rates of rad10? strains compared to wildtype and a 1.20 fold increase in rad1? strains. The increase in both strains was found to be insignificant using a two-tailed t test with p values of 0.063 and 0.052 for rad1? and rad10? strains respectively. While the results from the respiration loss assay indicate that RAD1 and RAD10 may be involved in maintaining the integrity of the mitochondrial genome, further exploration cannot support this claim. RAD1 and RAD10 are not involved in maintaining the mitochondrial genome through the single-strand annealing pathway it is known to function in within the nucleus. The genes may be involved in another pathway which was not tested in the assays used in this study such as nucleotide excision repair within the mitochondria.