DNA Repair in Yeast

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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.

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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.

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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!

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Planarians, eternally cute worms

Growth and development, including cell division and differentiation, are key parts of biology. Related to development and cell activity are the topics of healing and regeneration. We, as mammals, have the ability to heal most wounds with scarring, but humans can only regenerate our fingertips and liver. If we were faced with more substantial losses, there would be trouble. But planaria have no need to fear!

Planaria are flatworms of the phylum Platyhelminthes. Like all flatworms, they are acoelomates (have no body cavity). They live naturally as scavengers and predators to small protists at the bottom of ponds or streams and are sensitive to pollution and can only live in clean waters. In the lab, they live in petri dishes filled with spring water (Poland Spring® works nicely). Planaria asexually reproduce through fission, but as hermaphrodites, they are capable of sexual reproduction (it is very rare).

The two “eyes” on their triangular head gives planaria their adorable cross-eyed look. They are light-sensitive eyespots that help guide planarians away from light (negative phototaxis). They also use chemoreceptors on ciliated auricles, the ear-like extensions of the head. They have two ventrolateral and many transverse nerve cords that detect external stimuli and one muscular pharynx where food enters and waste leaves.

The reason planaria are so extensively used in the lab is due to their insane regenerative ability. Cut a planarian in half, and in a few weeks, you’ll have two healthy planaria (but abnormal regeneration is possible). In fact, a planarian can regenerate from as small as 1/270th of a body piece. Thus, a planarian seems to be immortal because it has an indefinite ability to regenerate its cells. This ability is due to neoblasts (totipotent adult somatic cells) that are able to replace cells during regeneration and wound healing with the formation of the blastema, an accumulation of neoblasts. Polarity and other factors that guide the regeneration process have been investigated and debated for years. 

For those that are concerned, planaria naturally use this regeneration process during reproduction (fission), where they tightly adhere their posterior end to a substrate and pull forward to rip themselves in two. While mutilation of an animal should never be taken lightly, scientists are doing nothing more than what the animal does to itself.

For more information: basic facts about planaria, mini documentary

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!

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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)