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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Cell Division

Why do cells divide?

The continuity of life is based upon the reproduction of cells. The single-celled amoeba divides to produce an entirely separate organism, while multicellular organisms such as humans arise from single cells via cell division. Furthermore, cells are not immortal and must be renewed, as seen in the creation of new red blood cells in bone marrow. Thus, there are 3 purposes for cell division: reproduction, growth and development, and tissue renewal or repair.

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Basic overview of Mitosis

Eukaryotic cell division consists of mitosis (the division of the nucleus) and cytokinesis (the division of the cytoplasm). FullSizeRender 84

FullSizeRender 86Interphase: cell growth and copying of chromosomes in preparation for cell division
  • First Gap (G1) represents the first part of interphase during which the cell grows and functions normally.
  • DNA begins to replicate and cytoplasm increases in Synthesis (S).
  • In Second Gap (G2), the cell checks for DNA damage that may have occurred during S phase and will proceed to Mitosis (M) phase once all necessary proteins are present.
  • Centrosomes duplicate, each with two centrioles.
  • Chromatin has not condensed yet and is not visible. The nuclear envelope and nucleolus are still present.
Prophase: The first step in Mitosis
  • Chromatin fibers condense into discrete chromosomes, each with 2 sister chromatids.
  • The nucleolus disappears.
  • Mitotic spindle (microtubules) begins to form.
Prometaphase: Chromosomes released from the nucleus
  • Nuclear envelope disintegrates.
  • Microtubules from the centrosome attach to the chromosomes via the kinetochore (on the centromere).
Metaphase: Chromosomes line up
  • Centrosomes are each at opposite poles.
  • Chromosomes are guided by the microtubules to the metaphase plate at the equator of the cell.
Anaphase: The shortest stage of Mitosis
  • The two sister chromatids separate and migrate to opposite poles.
Telophase and cytokinesis: two identical daughter cells are formed
  • Nuclear envelope and nucleoli reappear to enclose each group of chromosomes.
  • The cell splits into two new cells.
  • Cleavage furrow is formed in animal cells; cell plate in plants.

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Prokaryotes (bacteria and archea) reproduce by binary fission, in which the chromosome simply replicates and the two chromosomes actively move apart to form two identical organisms. Remember, prokaryotes have no nucleus and have a single circular chromosome that contains their genetic information.

Controlling Cell division

While cell division is an essential part of life, it would be disastrous of cells had no way of regulating this process.

  • The cell cycle is regulated largely by the accumulation of cyclin that combines with cyclin-dependent kinase to form MPF. The G2 checkpoint is only passed when there is a sufficient amount of MPF.
  • Platelet-derived growth factor (PDGF) promotes cell division by allowing the cell to pass the G1 checkpoint and begin preparing for mitosis. This is used by platelets to clot blood.
  • External factors also play a role. Density-dependent inhibition prevents cells from overcrowding, allowing them to only grow as a single layer. Cells may also exhibit anchorage dependence in which they will only divide if attached to a substrate such as tissue.

The loss of control of the cell cycle cause cells to divide abnormally, forming tumors. These cancer cells have no means of regulating cell division and will multiply rapidly.

The ability to reproduce and regulate cell division is essential for the continuity of life.

Sources: Campbell Biology 9th Edition

Making Bacteria Glow

Bacteria are used in the lab because they are inexpensive and easy to take care of. E. coli (nonpathogenic strain) is a common strain of bacteria that you may encounter in experiments, especially those dealing with transformation. In addition to their main chromosomal DNA, bacteria have another (physically separate) circular piece of DNA known as the plasmid. The plasmid is often manipulated in the lab (using various restriction enzymes) so that the bacteria is able to take up the modified plasmid and become a host organism that can produce the desired protein.

When learning about plasmids and bacterial transformation, this experiment is one of the most common experiments that done in the lab as an educational tool. If you’re interested in biotechnology or any biological laboratory science, you’ll definitely come across this experiment. (I’ve done it three times already!)

Results

Transformed E. coli under UV light

Transformed E. coli under UV light

Since future generations of bacteria will also express the inserted gene, this method is usually the first part of the upstream process in biopharmaceutical production. For example, if you wanted to collect a large amount of the GFP protein, you would use this to collect bacteria that produced GFP, and then use bioreactors to grow even larger cultures of it.

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)