In vitro Reconstitution of DNA Segregation; A Plasmid Spindle Driven by a Dynamically Unstable Actin Homologue

The par operon of the R1 plasmid constructs a DNA segregating spindle from three components, parC, ParR, and ParM. parC is a centromeric stretch of DNA which is bound by the protein ParR. ParM is an actin homologue that polymerizes into two stranded filaments that can interact with the ParR/parC complex. These three components are both necessary and sufficient to segregate parC containing plasmids, implying that the par operon is a biochemically self-contained DNA segregation apparatus. To elucidate how this system functions to segregate plasmid DNA, we wished to determine the intrinsic kinetic properties of ParM assembly, and how this assembly is regulated by the interaction with its cargo, the ParR/parC complex.We find that ParM displays three properties that distinguish its assembly from that of eukaryotic actins. 1) ParM displays hydrolysis dependent dynamic instability, the stochastic switching between states of growth and rapid depolymerization. 2) ParM filaments display a very rapid rate of nucleation, occurring at a rate 200-fold faster than actin. 3) ParM filaments exhibit no kinetic polarity, with equal rates of growth at each filament end.By combining ParM with bead immobilized ParR/parC we demonstrate that these three components can create bipolar spindles in vitro similar to those observed in vivo. ParR/parC coated microspheres are connected by bundles of ParM filaments that elongate over time, pushing the microspheres apart over long distances. Speckle microscopy and photobleaching experiments demonstrate that this motility is driven by insertional polymerization at the bead surface. We find that the ParR/parC complex prevents ParM filament ends from catastrophe by stabilizing filaments down to the ATP-ParM critical concentration. Futhermore, we show that the dynamic instability of ParM is critical to the spatial regulation of the R1 spindle, as filament ends that are not bound at each end by ParR/parC are destined to undergo catastrophe. The dynamic instability of ParM also provides an energetic differential to power spindle elongation, as the turnover of background, unbound filaments provide the monomer excess required for the growth of stabilized, spindle associated filaments