ParB spreading on DNA requires cytidine triphosphate in vitro.

Abstract

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-<i>parS</i> system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on <i>parS</i> sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at <i>parS</i> sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a <i>parS</i>-dependent ParB spreading event using purified proteins from <i>Caulobacter crescentus</i> and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-<i>parS</i> with ParA and SMC.