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For all organisms, segregation and proper intracellular localization of DNA are essential processes in ensuring faithful inheritance of genetic material. In prokaryotes, several different mechanisms have developed for efficiently moving chromosomal DNA to proper cellular locations prior to cell division, and the same holds true for bacterial plasmids. Low-copy-number plasmids and bacterial chromosomes encode active partition systems to ensure their inheritance within a bacterial cell population. One of the well-studied models of partition is that of the P1 plasmid in E. coli. The partition system encoded by the P1 plasmid is known as parABS - ParA is the partition ATPase, ParB is the partition site binding protein and parS is the partition site. The goal of this thesis was to investigate the nucleoprotein complexes essential in the P1 plasmid partition reaction. First, I examined how a single ParB dimer can bind its complicated arrangement of recognition motifs in parS to initiate the partition reaction. I then characterized a novel ParA interaction with the host nucleoid that is critical for proper P1 plasmid dynamics in vivo. Finally, I demonstrate how ParA can act as an adaptor between the nucleoid and the partition complex; effectively allowing the plasmid to use the nucleoid as a track for its intracellular movement and localization. My thesis work provides evidence towards a model that explains the P1 plasmid partition mechanism.

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The accurate distribution of genetic information (chromosomes) during cell division is essential for the growth and proliferation of all living organisms. Errors in chromosome segregation in humans have been linked to cancer progression, infertility and developmental diseases. In my PhD I study how chromosome segregation is regulated in the genetically amenable budding yeast Saccharomyces cerevisiae. Since the mechanisms of chromosome segregation are highly conserved amongst eukaryotes, studies in yeast will provide a fundamental understanding of this process. Sgo1 is the budding yeast member of a highly conserved family of shugoshin proteins, which play a key role in chromosome segregation. My work characterizes a previously unidentified role of Sgo1 in inhibiting separase; an enzyme that triggers chromosome segregation by cleaving the cohesin protein complex that holds replicated chromosomes together. I demonstrate that this novel function of Sgo1 requires a specific form of Protein Phosphatase 2A (PP2ACdc55), an enzyme that itself is highly conserved amongst eukaryotes. I propose that PP2ACdc55 is a separase inhibitor that is employed by Sgo1 when sister chromatids are not under tension. Finally, I go on to initiate preliminary studies into the mechanism whereby PP2ACdc55 inhibits separase. In sum, this study uncovers an additional layer of separase regulation mediated by Sgo1 and PP2ACdc55 and therefore makes a significant contribution to our understanding of the all-or-nothing nature of chromosome segregation.