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DNA repair enzymes in Prochlorococcus marinus strain MIT9312

Prochlorococcus marinus, a marine cyanobacterium, is one of the most abundant photosynthetic organisms in the ocean. P. marinus contributes towards approximately 8.5% of global ocean primary productivity. P. marinus is of interest because of the cell’s simple and unique photosynthetic system, efficient carbon concentrating mechanism, and streamlined genome. P. marinus strain MIT9312 is a high-light adapted strain that thrives in the upper euphotic zone where there are minimal nutrients and high levels of UV exposure, which would typically put an organism at risk. Understanding the function of repair proteins is significant for being able to identify the DNA repair pathways used by Prochlorococcus to survive under increasingly more harmful conditions. My research, outlined in this thesis, aimed to characterize DNA repair enzymes in Prochlorococcus marinus strain MIT9312 to further our understanding of cyanobacterial growth, survival, and repair. LigW is an ATP-dependent ligase that is unique to strain MIT9312. It was found to have low ligase activity on nicked DNA compared to other ligases, making its retention in the small Prochlorococcus genome unusual. It is suspected that LigW could achieve higher ligation rates by interacting with other proteins. LigW is in an operon with other proteins that are predicted to promote LigW activity or have their own DNA modifying activities. The three proteins of interest have been annotated as Pmar3, Pmar4, and Pmar5. The individual function and relationships between each protein were investigated in this thesis. This thesis focused on recombinantly producing and biochemically characterizing three of the proteins adjacent to LigW and assessing their activities in vitro. Pmar3 was successfully expressed, purified, and characterized as a potential nuclease. Further mutagenesis work will confirm the proposed function of Pmar3. Attempts were made to characterize Pmar4, with further optimization of expression and purification protocols necessary. Pmar5 has been characterized as a pentameric Mg-dependent topoisomerase-like protein, with an inability to re-join DNA. It is likely that these proteins on the LigW operon work together with LigW in a currently unidentified repair pathway. Once the LigW DNA repair system is characterized, then this can be more easily identified and understood in other organisms where it may not be detected at present.
Type of thesis
The University of Waikato
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