Structural studies of the Vibrio cholerae chromosome segregation system

The efficient segregation of replicated genetic material is an essential step for cell division. In eukaryotic cells, sister chromatids are separated via the mitotic spindles. In contrast, bacterial cells use several evolutionarily-distinct genome segregation systems. The most common of these is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Using the human pathogen Vibrio cholerae, which possesses two chromosomes each encoding its own Par system, I first determined a purification protocol for its ParA proteins (ParA1 and ParA2, respectively). I then used negative-stain TEM to investigate the oligomerization of ParA2, in the presence of nucleotide as well as DNA. I also determined its crystal structure, in both apo and ADP-bound states. Finally, I used cryo-EM to determine its structure bound to DNA, the first structure of a ParA filament. Collectively, these structures offer insight into its conformational changes from dimerization through to DNA binding and filament assembly. Specifically, it is shown that the ParA dimer is stabilized by nucleotide binding, and forms a left-handed filament using DNA as a scaffold. The structural analyses also reveal dramatic structural rearrangements upon DNA binding and filament assembly. Finally, through negative-stain electron microscopy, I show how filament formation is controlled via C-terminal basic residues of ParA2, with nucleotide binding and hydrolysis being key to filament assembly and disassembly along DNA. The data reported here provides the structural basis for ParA's cooperative binding to DNA and the formation of high ParA density regions on the nucleoid, and suggest a role for its filament formation in DNA segregation in bacteria.