Kinetic pathway of ATP-induced DNA interactions of ParA2, a protein essential for segregation ofVibrio choleraechromosome 2

ABSTRACT Vibrio cholerae chromosome 2 (Chr2) requires its own ParABS system for segregation. Without it, V. cholerae becomes nonviable and loses pathogenicity. ParA2 of Chr2 is a Walker-type ATPase that is the main driver of Chr2 segregation. Most of our understanding of ParA function comes from studying plasmid partition systems. How ParA provides the motive force in segregation of chromosomes, which are much larger than plasmids, is less understood and different models have been proposed. Here we analyzed in vivo behavior and kinetic properties of ParA2 using cell imaging, biochemical and biophysical approaches. ParA2 formed an asymmetric gradient in the cell that localized dynamically in the cell cycle. We found that ParA2 dimers bind ATP and undergo a slow conformational change to an active DNA-binding state, similar to P1 ParA. The presence of DNA catalyzes ParA2 conformational change to allow cooperative binding of active ParA2 dimers to form higher-order oligomers on DNA. Nucleotide exchange rates were also slow, thus providing a control of ParA2 recruitment and dynamic localizations. Although highly conserved in biochemical properties, ParA2 showed faster overall ATP cycling and DNA-rebinding rates than plasmid ParAs, suggesting that this could be shared kinetic features among chromosomal ParAs to regulate the transport of a much larger DNA cargo.