<p>DNA partition or segregation is the process that ensures the stable inheritance of genomic material. The majority of the bacterial plasmid and some chromosomal partition systems utilize ParA Walker-box-based partition systems. These systems require three components: a DNA centromere site, the ParA ATPase, and the ParB centromere binding protein. ParB binds to the centromere to form the partition complex, which then recruits the motor protein ParA. ParA mediates the partition of replicated DNA by a still poorly understood mechanism. Notably, recent data indicates that ParA Walker-box-based partition systems are employed not only by bacterial plasmids and chromosomes but also DNA elements in archaea. The work in this thesis focused on a homolog of the ParA protein from the first identified archaeal plasmid partition system, located on the plasmid pNOB8. pNOB8 plasmid is harbored in the thermophilic archaeaon, Sulfolobus solfataricus. The goals of this work were to structurally and biochemically characterize the ParA homolog to gain insights into its function.</p><p>Towards these goals, the structure of the ParA homolog was solved by X-ray crystallography in its apo and ADP bound states to resolutions of 2.45 Å and 2.73 Å, respectively. The overall structure was similar to bacterial ParA proteins. We next demonstrated that, similar to bacterial ParA proteins, this ParA homolog harbored ATP-dependent nonspecific DNA capabilities by using fluorescence polarization based DNA binding assays. By mutating the residues in the deviant Walker A motif, we were able to demonstrate the importance of ATP binding in its DNA binding function. Moreover, characterization of ATP and ADP binding were performed using ITC. Finally, we observed that ParA was able to form polymers in the presence of ATP, using negative stain electron microscopy. Our findings provide evidence that ParA Walker-box-based partition systems, which are the most common systems in bacteria, appear to also be found in archaea.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/9923 |
| Date | January 2015 |
| Creators | Lee, Jeehyun |
| Contributors | Schumacher, Maria A |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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