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DNA replication is a process that is vital to cell survival. When double stranded DNA goes through the replication process, it splits into two separate strands with the help of a helicase. When a cell in the process of replicating its DNA encounters DNA damage, the replication proteins fall off the DNA strand and DNA replication stops; in other instances cells can utilize the replication restart pathway. This pathway allows certain cells to overlook damaged DNA strands, reload replication proteins back on the replication strand, and proceed with replication. Deinococcus radiodurans is one organism that is able to utilize this pathway despite being exposed to high levels of radiation. It is believed that certain replication proteins including polymerases, primases, and helicases are used to help an organism use the replication restart pathway. DnaB is a replicative helicase that is responsible for unwinding the majority of the double stranded DNA on a chromosome, SSB prevents the separated DNA strands from reannealing, and PriA is the most important protein in the replication restart process because it is what initiates the process of reloading the proteins on the DNA strand. The goal of this project was to determine if the PriA protein is able to reload the DnaB helicase onto the DNA replication fork, specifically looking at whether DnaB was loaded with no leading strand gap on the DNA strand. Through transforming these D. Rad proteins into E coli cells, synthesizing and purifying these proteins, creating a DNA fork to unwind, and then performing helicase assays to test the unwinding capabilities of the DNA replication fork, we were able to test our hypothesis. Our results indicated that no DNA unwinding occurred on the DNA fork. This result can mean one of several things: incorrectly synthesized proteins or the DnaB helicase was not able to be reloaded on the DNA fork.
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"Investigating Survival Strategies of a Radioresistant Bacterium: Deinococcus Radiodurans" (2014). Stander Symposium Projects. 467.
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