Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS

Abstract

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTP{gamma}S. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of a parS loading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg2+ complexes move along DNA following loading at parS sites and protein:protein interactions result in the localised condensation of DNA within ParB networks.