INVESTIGATING THE MECHANISTIC FEATURES OF ParABS-MEDIATED Vibrio cholerae CHROMOSOME 2 SEGREGATION

In all cell types, stable generational viability is dependent on the faithful inheritance of
genomic material in daughter cells. This entails replicated DNA being segregated to opposing
sides of the cell prior to cell division at mid-cell. In bacteria, a minimal ParABS (partitioning)
system is used to correctly localise DNA cargos and is found on the chromosomes of up to
70% of species. A centromere-like site, parS, is bound by the CTP-binding protein, ParB. An
ATPase with DNA-binding activity, ParA, acts as a motor protein to drive segregation of the
ParB-parS complex, along with the attached DNA cargo. The underlying mechanism has been
derived mostly from studies on plasmid Par systems. However, despite recent progress for
Par-mediated chromosome segregation, the exact mechanism(s) of their action have yet to
be deciphered. Vibrio cholerae, the causative agent of cholera, has two chromosomes, with
one using a Par system closely related to those found on plasmids, making it an ideal
chromosomal Par model to study. The goal of this thesis was to biochemically characterise
the ParABS system of V. cholerae chromosome 2. First, each step in the ATPase cycle of
ParA2 was examined and it was shown that there are key differences to plasmid ParA
proteins, with an overall faster ATPase cycle likely responsible for driving translocation of a
much bigger DNA cargo. Secondly, the formation of the ParA2-ParB2-parS2 complex was
investigated, and dynamic self-assembly was seen in the presence of ATP and CXP.
Throughout the study, findings were compared with known plasmidal and chromosomal Par
systems in order to contribute to a greater understanding of the mechanism(s) of bacterial
chromosome segregation.