Ancestral sequence reconstruction reveals CTP-dependent parS binding as an ancient feature of ParB DNA segregation proteins

In most bacteria, chromosome and low-copy plasmid segregation are mediated by the ParABS system. Its component ParB functions as a DNA sliding clamp that assembles on centromere-like parS sites to form large nucleoprotein complexes, which are subsequently positioned by the ATPase ParA. Loading of ParB onto DNA is regulated by a recently discovered, conserved CTPase domain, yet the evolutionary origin of this regulatory module remains unclear. Here, we apply ancestral sequence reconstruction to resurrect anci¬ent ParB proteins dating back to the last common ancestor of bacteria. Biochemical, structural, and cell biological analyses demonstrate that these reconstructed proteins display all core activities of their mod¬ern counterparts, suggesting that the ParABS system emerged early during bacterial evolution and has essentially re¬mained unchanged ever since. More broadly, our findings indicate that regulatory CTP¬ases repre¬sent an ancient molecular innovation, whose origins can be traced back to the earliest stages of life.