ABSTRACT
In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS 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 parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus 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-parS with ParA and SMC.
Footnotes
1) Additional experiments: Figure1-figure supplement 1, Figure 3-figure supplement 2, Figure4-figure supplement 1, Figure 6, and Figure 6-figure supplement 2. 2) Updated Introduction, Materials and Methods, Results, and Discussion sections. 3) Mis-labelling from the original version: Morella thermoacetica His6-MBP-ParB (in the original manuscript) should be Dechloromonas aromatica His6-MBP-ParB, and 0.01 mM CTP was used for Zymomonas mobilis and Xanthomonas campestris ParB instead of 1mM CTP used for all other ParB samples. These two mislabellings have been corrected and the figure legend updated in the revised manuscript. The conclusion that ParB-CTP interaction with DNA is most likely conserved among ParB orthologs stands.
