DNA damage is often responsible for many types of diseases such as cancer. In cells, the most common type of DNA damage is the double strand break (DSB). Cells have many rescue methods to repair their broken DNA. This summer, I worked at a lab and helped a postdoc on her project in studying the role of sumoylation in promoting DNA checkpoint in yeast.
In summary, once a DSB is detected, a cascade of proteins are recruited, as shown in the diagram above. One result of the DSB repair process is cell cycle arrest, also known as the checkpoint response. While the cell cycle is arrested, the cell initiates homologous recombination, which is the main repair mechanism for a DSB. This is when sumoylation of the common protein, RPA occurs.
There is evidence of a link between the checkpoint response and the sumoylation of RPA. By creating many yeast strains with varying genotypes, we study how the sumoylation of RPA is involved in the checkpoint response.
I went through the process shown to the left for each strain. Beginning with mating, the entire process takes about a week. At first I experienced some trouble with primers for PCR, and with my mentor’s help, we troubleshooted our process. Within a week, we managed to fix the issue and come out with beautiful gels. This was a valuable laboratory experience for me since I learned how to go about analyzing and correcting my methods. By the end of the summer, I built about 12 different strains that will be useful in understanding the significance of sumoylation in DNA damage checkpoint.
The most tedious but one of my favorite parts of this experience was learning how to dissect yeast tetrads. I spent hours at the dissection microscope carefully separating each of the four clumped spores and placing them carefully in rows on the plate. The next few days I would watch them grow into neat little rows of colonies, and then proceed to select and analyze each colony.
This experience allowed me to thoroughly explore the processes of homologous recombination and the DNA checkpoint in a model organism, yeast. I was excited to have such an excellent opportunity to study and understand the unique proteins involved in the steps of DNA repair firsthand. As I learned each of their roles, I became more and more fascinated in the complexity of the cell’s processes. With this enthusiasm, I look forward to studying biology in the future.
Click below to see more of my notes!