DNA Repair in Yeast

IMG_9087

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.

Screen Shot 2017-11-19 at 6.09.39 PM

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.

Screen Shot 2017-11-19 at 6.10.03 PMI 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.

IMG_9083

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!

Continue reading

A Study in Yeast

Yeast Life cycle

Yeast are simple unicellular eukaryotes that undergo the biochemical process of fermentation, releasing ethanol and carbon dioxide as waste products. We can all thank yeast for our bread and beer, but there’s much more to this little organism.

A Study in Yeast

Yeast (S. cerevisiae) is a widely used model organism in the laboratory because its genome, which is completely sequenced, has many similarities to humans. Yeast, as eukaryotes like our cells, have chromosomes packaged as DNA inside a nucleus and undergo similar biochemical pathways (ex. DNA repair, cell cycle). Despite the drastic physical differences, they have hundreds of “swappable” and homologous genes to humans and thus, proteins with similar biochemical properties as humans, making them ideal for research studies on disease (neurodegenerative diseases, cancer). Not only that, yeast are very easy to care for in the lab; they grow in colonies in liquid or solid media (agar), do not require oxygen (but grow faster with oxygen), and can grow well at room temperature (optimal temp. 30ºC). I started with a single swipe of yeast cells on my plate, and after one night, the plate was filled!

Yeast

I did an experiment based on their mechanisms for DNA repair using UV light. I loved working with yeast because it smells like bread (I just caught a few whiffs when I opened up the dish, please don’t deliberately sniff the yeast).

Fun fact: The mating projections of yeast are called shmoos!

 

For more information: basic outline on why we use yeast in research, “Could Yeast be the New Hero in Biomedical Research?”, PDF on yeast and neurodegenerative diseases (first few pages are helpful)