| The ParM protein, encoded by the parMRC locus from plasmid R1, is the best-characterized actin homolog present in prokaryotes. In conjunction with a nucleoprotein complex formed by a DNA binding protein ParR and a DNA centromere parC, ParM constructs a simple mitotic spindle that pushes plasmid DNA to opposite poles of a bacterial cell for DNA segregation. Like actin, ParM assembles into ATP-dependent double-stranded, helical filaments via a nucleation condensation reaction. However, unlike actin, it displays three kinetic properties that are essential to its function: rapid spontaneous nucleation, bilateral elongation and ATPase dependent dynamic instability: the stochastic switching between filament elongation and rapid catastrophic shrinkage. In this dissertation, we investigated three questions related to ParM's evolution and function. First, many divergent R1 ParM homologs are encoded by other plasmids, and function in plasmid DNA segregation. We asked whether the filament architecture and assembly dynamics are conserved in a divergent member of the ParM family, the ParM protein from plasmid pB171. Using microscopic and biophysical techniques, we compared and contrasted the architecture and assembly of these related proteins. Despite being only 41% identical, we find that R1 and pB171 ParM polymerize into nearly identical filaments that display similar assembly dynamics, suggesting that structure and function are conserved in the ParM family. Second, we asked whether ParM prefers ATP or GTP in vitro. Using biochemical assays and TIRF microscopy, we find that ParM displays similar polymerization kinetics and stability in the presence of either nucleotide, but binds to ATP ten times tighter than GTP. Third, the structural basis for ParM filament dynamic instability remains poorly understood. Using cryo-electron microscopy to evaluate ParM filaments, we find that the nucleotide binding pocket of ParM protomers exist in an open or closed state, and that ATP hydrolysis shifts the distribution of protomers states; these findings suggest a mechanism for dynamic instability. |
