Essays on Science and SocietyGE PRIZE ESSAY

Understanding a Minimal DNA-Segregating Machine

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Science  05 Dec 2008:
Vol. 322, Issue 5907, pp. 1486-1487
DOI: 10.1126/science.1168506

GE Healthcare and Science are pleased to present the prize-winning essay by Ethan Garner, a regional winner from North America, who is the Grand Prize winner of the GE & Science Prize for Young Life Scientists.

A current challenge in biology is to bridge the gap between the parts and the whole, to reconcile the biochemical properties of individual proteins with the emergent behaviors of multipart molecular machines. In theory, if every kinetic rate constant for every interaction were measured, we could gain a complete mechanistic understanding of a biological process. Because the number of required measurements scales with the number of components, most complex biological systems present difficult challenges for this approach. However, when quantitative insight is matched with the proper system, the goal of elucidating the biochemical basis of emergent behaviors can be realized. For example, to gain a molecular-level understanding of DNA segregation, a fundamental, mesoscale process, I turned to a system that has been forced to be minimal and self-contained during its evolution: low-copy bacterial plasmids. To maintain their inheritance against the fitness costs imposed by their extra metabolic burden, these exogenous elements have evolved minimal, self-contained, DNA segregation machines.

At a minimum, a DNA-segregating system needs to accomplish three tasks. First, it must count the copies of DNA to know when to initiate segregation. Second, it must exert directional force to propel these DNA copies away from each other. Third, this system must be spatially aware of the cellular geometry so that the DNA is propelled toward each eventual daughter cell. For the Escherichia coli R1 drug resistance plasmid, all of these tasks are conferred by the par operon, which constructs a mitotic spindle out of only three components (1). The centromeric sequence parC contains 10 repeats that are bound by the adapter protein ParR. This ParR/parC complex interacts with ParM, a distant actin homolog that polymerizes into helical filaments (2). In my thesis work, I determined how these three components interact to accomplish the systems-level task of DNA segregation. To elucidate this mechanism of plasmid segregation, I needed to understand the nature of ParM polymerization, how the ParR/parC complex affected ParM filaments, and finally, how these three components interact to push the plasmids to the poles of the cell.

First, I conducted a complete characterization of ParM assembly dynamics, measuring all available rate constants for this polymer (3). Although ParM is a structural homolog of eukaryotic actin, I found three distinct differences between these polymers. First, ParM nucleates filaments at a rate 300 times as fast as actin. Second, unlike any other previously observed polymer, ParM shows no polarity as it grows at equal rates at each end of the filament. The most striking difference between ParM and actin is that ParM exhibits dynamic instability, the stochastic switching between states of growth and rapid disassembly (4). Previously, this behavior had only been observed for eukaryotic microtubules. ParM's dynamic instability is driven by ATP hydrolysis, as ADP-bound ParM filaments display a much higher dissociation rate than the ATP-bound filaments.

Dynamic instability and bipolar stabilization drive plasmid segregation.

The ParR/parC complex can capture cytoplasmic ParM filaments. Filament ends bound by ParR/parC are stabilized (blue) while unbound filaments (red) and destined to undergo catastrophe. A productive spindle is formed when both ends are bound by ParR/parC. Turnover of the unattached, background filaments provides the energy differential (arrows) to power spindle elongation.

The combination of dynamic instability and rapid nucleation causes solutions of ParM to consist of a population of short, dynamic filaments that nucleate and turn over throughout the volume. This unstable, transient nature appears at odds with the formation of a rigid force-generating mitotic spindle, suggesting that the ParR/parC complex must alter ParM filament dynamics. I tested this hypothesis by reconstituting the par system from purified components (5), combining parC-conjugated beads with ParR and fluorescently labeled ParM. Isolated parC beads displayed short, dynamic asters of ParM emanating from their surface, as if they were searching the surrounding volume. When two parC beads came into close contact, stable bundles of filaments formed between the beads. These filaments then elongated at a constant rate, pushing the beads in opposite directions. Creating fiducial marks on these filaments demonstrated that they elongate at equal rates at each bead surface, indicating that new monomers are added into the spindle through a process of insertional polymerization at the ParR/parC complex. This was the first in vitro reconstitution of a DNA-segregating system from purified components.

The fact that we observed long, stable ParM filaments only between pairs of beads indicated that the filaments are stabilized against catastrophic disassembly when bound at both ends by ParR/parC.When these spindles were severed with laser irradiation, these filaments would rapidly depolymerize. This bipolar stabilization provides an intrinsic counting mechanism, as productive and sustained filament elongation only occurs between parC pairs.

I then tested whether these spindles could locate the ends of a volume by confining them in microfabricated channels of various shapes. These spindles aligned with and elongated along the long axis of these spaces, indicating that this simple system is sufficient to find the long axis of a cell.

In addition to demonstrating that these three components are necessary and sufficient to generate the emergent behaviors required for DNA segregation—counting, force generation, and spatial awareness—this work also elucidated the biophysical relationship between dynamic instability and force generation. By conducting spindle assembly assays at varying concentrations of ParM, I found that filaments bound at each end by ParR/parC behave as if the entire filament is composed of the higher-affinity, ATP-bound polymer. In essence, this stabilizes the bound filaments to a lower energetic level than the unbound filaments that turn over in solution (see the figure). This indicates that the dynamic instability of the unattached filaments provides the monomer excess that powers the elongation of the stabilized ParR/parC attached filaments.

These studies provide a framework for understanding the essential principles of DNA segregation. Furthermore, they demonstrate that biology can solve complex tasks with a surprisingly small number of components. This minimal solution to DNA segregation is not an isolated case; many low-copy plasmids have independently converged on three-component systems by co-opting a variety of different polymers from their hosts (6-9). Kinetic dissection of a range of these self-contained mitotic machines may uncover the differing solutions that biology has evolved to ensure genetic inheritance and thus broaden our understanding of this fundamental biological process.

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