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-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.

Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities

“…Recent evidence has added a new nucleotide cofactor to partition with the demonstration that ParB binds and hydrolyses CTP (43)(44)(45). Further, the evidence indicates that CTP, which binds to the N-terminal domain of ParB, allows ParB to clamp onto DNA when it loads at parS, and facilitates loading multiple ParB molecules.…”

Spatial control over near-critical-point operation ensures fidelity of ParABS-mediated bacterial genome segregation

“…Such a study is also be complicated with the transient interactions between ParA and ParB that both exist in different biochemical states Ah-Seng et al 2009). The recent findings that ParB binds CTP (Soh et al 2019;Osorio-Valeriano et al 2019;Jalal, Tran, and Le 2020) and that parS-mediated CTP hydrolysis stimulates ParA interaction (Osorio-Valeriano et al 2019; Jalal, Tran, and Le 2020) could open interesting molecular clues to control this interaction. Moreover, our model describes the PC partitioning along the nucleoid surface as a 2D problem.…”

Bacterial chromosome segregation by the ParABS system

“…Fluorescence recovery after photo-bleaching (FRAP) experiments have shown that ParB F molecules rapidly exchange between different clusters, further highlighting the dynamic nature of cages of ParB-DNA in vivo [70]. The nucleation and caging model has also been shown applicable to the Vibrio cholerae chromosomal ParB-parS system, suggesting that this dynamic self-assembly mechanism might be conserved from plasmids to chromosomes [ between the nucleation and translocation modes [52,71]. Mutant proteins (N112S and N172A of B. subtilis and M.…”

“…Recent studies have uncovered a new cofactor of ParB [ 51 , 52 ]. Various plasmid- and chromosome-encoded ParB and ParB-like proteins have been found to bind and hydrolyse cytidine triphosphate (CTP) to cytidine di-phosphate (CDP) and inorganic phosphate [ 51 , 52 , 71 ]. A co-crystal structure showed CDP binding to the arginine-rich patch at the NTD of B. subtilis ParB (CTP was hydrolysed to CDP during crystallization) [ 52 ].…”

“…A comparison between the B. subtilis ParB–CDP structure and the H. pylori ParB– parS structure suggested that CTP binding induces a conformational change at the central DNA-binding domain that is incompatible with parS binding [ 52 , 54 ]. Studies with C. crescentus and M. xanthus ParBs further showed that CTP binding reduces ParB nucleation at parS and/or liberates pre-bound ParB from parS [ 51 , 71 ], thereby facilitating the escape of ParB from a high-affinity nucleation site to a low-affinity neighbouring DNA. Therefore, CTP probably serves to switch ParB from a nucleating to a sliding mode ( figure 1 e ).…”