Presentation description

For my UROP project, I have been studying DNA repair enzymes found in microbes living at the Lost City, an exoplanet environment at the bottom of the Atlantic Ocean where there is no sunlight and very little oxygen, conditions that resemble a younger Earth with the importance of understanding the origin of life. Recent work from the Horvath and Brazelton labs discovered genes encoding the DNA repair enzyme MutY in the metagenomes from the Lost City. The enzyme MutY functions in suppressing mutations and therefore protects humans from developing cancer by finding and initiating base excision repair at 8-oxo-7,8-dihydroguanine (OG): Adenine sites. One of the Lost City MutYs seems to come from a microbe with unusual sulfur metabolism and is therefore of special interest. To explore how the DNA repair enzyme MutY adapted to the strange environment, I applied three biochemistry approaches: 1) molecular cloning to attach MutY-like enzymes to the soluble bacterial protein GST. GST is expected to improve the solubility of MutY and also make it easier to purify the protein, 2) protein expression tests to determine if soluble proteins are being expressed in different conditions, and 3) glycosylase assay to measure the enzyme activity. My cloning worked for four Lost City MutYs, and the proteins expressed well. One of these proteins appeared to be soluble and I am currently optimizing expressing conditions (temperatures and concentrations of IPTG) for this Lost City MutY from the sulfur metabolism microbe. My next step is to show that the protein can be purified and this will allow me to measure enzyme activity by the glycosylase assay at potentially extreme temperature and pH similar to the environment at the Lost City. Further studying the structural binding affinity of the enzyme's active site to a substrate and biochemical reaction catalyzed by MutY will help us better understand the inside environment of microbes and how organisms protect their genome from rapid mutation expected at extreme conditions.