Molecular insights into the mechanism of ParABS-mediated chromosome segregation

DNA segregation is an essential process in living organisms that ensures the physical distribution of genetic material to the nascent daughter cells prior to cell division. In most bacteria this process is mediated by the highly conserved ParABS system, which has been found to mediate the partition of bacterial chromosomes and low-copy plasmids. The system relies on interactions between two partitioning proteins, the CTPase ParB and the Walker ATPase ParA, as well as the origin-proximal centromeric sequence parS. After the start of chromosome replication, ParB dimers form CTP-dependent clamps on the parS sites and condense the replicated origin region into a large nucleoprotein partition complex that is subsequently transferred across the cell by the interaction of ParB with ParA-ATP dimers bound to the nucleoid. Although the function of the ParABS system has been studied intensively for the past 30 years the exact mechanism by which these interactions facilitate DNA segregation remains incompletely understood. In this work, we provide a comprehensive mechanistic analysis of the interactions within the ParABS system, by investigating the nucleotide-dependent interplay between ParB, ParA and parS at a molecular level in the model organism Myxococcus xanthus. To this end, we characterize interaction- and hydrolysis-deficient ParB and ParA variants using a broad range of methods, including biolayer interferometry, NMR spectroscopy, hydrogen-deuterium exchange mass spectrometry, enzymatic assays and in vivo localization studies. The results obtained uncover the determinants for the interaction of the two partition proteins and give insight into structural and catalytical mechanisms that ensure robust and directed segregation of their DNA cargo. Collectively, our work demonstrates the importance of CTP-dependent N-terminal dimerization of ParB for the interaction with ParA and identifies a previously unmapped ParB-binding region on the ParA dimer interface. Moreover, we show that the interaction with ParB increases the DNA-binding affinity of ParA, thereby promoting the formation of stable ParB-ParA-DNA complexes, which are then dissociated by the ParB-dependent stimulation of ParA ATPase activity. Lastly, we investigate the prevalence of ParB and ParA interaction determinants within orthologous systems, highlight a conserved mode of action between chromosomal and plasmid-derived (Type I) Par systems and define regions of ParB and ParA that can possibly be evolutionary modulated to either avoid cross-talk with analogous systems or adapt ParABS function towards the requirements of the specific organism. Collectively, our findings shed new light on a key aspect of ParABS function and thus significantly advance our understanding of the molecular mechanisms mediating prokaryotic DNA segregation.