The main aim of the project is to investigate the impact of mitochondrial processes on the survival of DNA damage response pathway mutants. This is useful to know as if it can be determined that a lack of functioning mitochondria affects the DNA damage response it could provide a convenient raison d'être for the Warburg effect. The Warburg effect being an altered metabolism seen in human tumours where cells have an increased uptake of glucose and a switch to glycolysis even when there is ample oxygen that would allow for the glucose to broken down for energy by oxidative phosphorylation. In the experiments Saccharomyces cerevisiae was utilised as a model. This was done because Saccharomyces cerevisiae can survive without its mitochondria, has protein homologues of the human DNA damage response proteins, and has a short generation time allowing for cultures to be performed relatively quickly.
There were two hypotheses that were tested. The first was that the removal of mitochondria directly impacts the function of the DNA damage response pathway lessening the impact of mutations in this pathway and thus promoting survival. The second, based on initial findings from spot tests, was that the removal of the mitochondria reduces the total amount of reactive oxygen species in the individual Saccharomyces cerevisiae cells and impairs apoptosis so that when proteins in DNA damage response pathway are mutated and generating reactive oxygen species the lower baseline amount of reactive oxygen species and the impaired apoptotic pathways prevent the death of the cell.
The first thing done in the project was the generation of Saccharomyces cerevisiae strains which lacked mitochondria, termed petite strains. This was done using ethidium bromide treatment. These strains were confirmed as petite using YPG plates as petite strains are unable to utilise glycerol as an energy source. Once confirmed as petite these strains were used in spot tests on plates containing drugs at various concentrations. These spot tests showed interesting improvements in survival on hydroxyurea containing media for mec1-4 and dun1Δ strains when petite. Also of note was the fact that there was no improvement in survival for the petite rad53-K227A strain. This is interesting as Rad53 lies in between Mec1 and Dun1 in the DNA damage response pathway which threw doubt on the first hypothesis explanation that the survival of the DNA damage response mutants is due to the loss of mitochondria affecting the functioning of DNA damage response pathway. The mec1-4 and dun1Δ strains also showed differences in their temperature sensitivities when petite. Overall, the results of the spot tests and previous studies led to the development of the second hypothesis that the survival was based on levels of reactive oxygen species.
The next thing which was looked at was the status of Rad53 and Sml1 in the petite and non-petite mec1-4 and wildtype strains when exposed to hydroxyurea. This was done by analysing Western blots for Rad53 and Sml1 from protein extracts. The results of these Western blots complicated the picture regarding the way in which the DNA damage response is impacted necessitating further research to find the reasons behind the apparent differences seen in the phosphorylation of Rad53 and Sml1.
The final experiment investigated whether there were significant differences in reactive oxygen species between untreated strains and hydroxyurea treated strains and between petite and non-petite strains. This experiment utilised the dye DCFDA which reacts with reactive oxygen species to form a fluorophore which was detected using a FACS machine. The results of this experiment suggested that there was no substantial difference in the levels of reactive oxygen species between the petite and non-petite mec1-4 strains.