ParB spreading on DNA requires cytidine triphosphatein 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.Faithful chromosome segregation is essential in all domains of life if daughter cells are each to inherit the full set of genetic information. The SMC-ParAB-parS complex is widely employed for chromosome segregation in bacteria 1-13 . The centromere parS is the first DNA locus to be segregated following chromosome replication 8,9,14,15 . ParB specifically nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo [16][17][18][19][20][21][22][23] . This nucleoprotein complex recruits SMC to disentangle and organize replicated DNA 3,11,13,24,25 . ParB-parS also interacts with an ATPase ParA to power the segregation of replicated chromosomes [26][27][28][29][30] . Engineered strains harboring a nucleation-competent but spreading-defective mutant of parB are either unviable 10 or have elevated number of anucleate cells 4,7,8,15,[31][32][33][34] . Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high molecular-weight nucleoprotein complex is not fully understood.Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 . The nucleation of ParB on parS has also been demonstrated in vitro 4,10,16,17,20,[37][38][39] , however parS-dependent ParB spreading has resisted biochemical reconstitution [17][18][19]40,41 . Unsuccessful attempts to reconstitute spreading in vitro suggests that additional factors might be missing. Recently, works by Soh et al (2019) and Osorio-Valeriano et al (2019) on Bacillus subtilis and Myxococcus xanthus ParB, respectively, showed that ParB binds and hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP), and that CTP modulates the binding affinity of ParB to parS 42,43 . A co-crystal structure of Bacillus ParB with CDP and that of a Myxococcus ParB-li...

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

“…CTP hydrolysis is not required for loading but might instead assist ParB recycling and control ParB spreading, as shown in vitro for C. crescentus ParB [140]. Spreading may also be restricted by road blocks formed by NAPs [122,140]. The role of CTP binding and hydrolysis in ParB-driven partition complex formation was also shown for M. xanthus ParB [141].…”

“…The steric hindrance between parS bound HTH motifs was suggested to promote the detachment of the ParB dimers in a closed conformation from parS, as well as sliding away (spreading). CTP hydrolysis is not required for loading but might instead assist ParB recycling and control ParB spreading, as shown in vitro for C. crescentus ParB [140]. Spreading may also be restricted by road blocks formed by NAPs [122,140].…”

“…The observation that ParB loading at parS and sliding on DNA requires CTP binding [122,140,141], a key metabolite in DNA and phospholipids synthesis, adds CTP to the players possibly regulating the ParB-DNA interactions. The motif containing amino acid residues responsible for CTP binding and ParB CTPase activity is one of the most conserved elements within the ParB protein sequences [122,141].…”

Distinct activities of bacterial condensins for chromosome management inPseudomonas aeruginosa

“…The ParB DNA-binding protein recognizes its target parS, present in one or several copies in the Ori region of bacterial chromosomes (Livny et al, 2007). ParB is a CTP hydrolase whose activity is critical for its interaction with parS (Jalal et al, 2020;Osorio-Valeriano et al, 2019;Soh et al, 2019). The resulting ParB/parS nucleoprotein complex encompasses several kbs of DNA and interacts with the ParA ATPase that drives its segregation (Kawalek et al, 2020).…”

Fine-tuning of the Smc flux facilitates chromosome organization inB. subtilis

“…In most bacteria, Smc-ScpAB complexes start DNA translocation from predefined entry sites, the 16-bp parS DNA sequences, which are generally found near the replication origin and are specifically recognized by the Smc-loader protein ParB (Gruber & Errington, 2009; Sullivan et al, 2009). ParB dimers form DNA clamps that self-load onto DNA at a parS site (Jalal et al, 2020; Osorio-Valeriano et al, 2019; Soh et al, 2019). As Smc complexes translocate away from the parS loading site in both directions (two-sided), they co-align the left and the right chromosome arms that flank the replication origin (Minnen et al, 2016; Tran et al, 2017; Wang et al, 2015, 2017).…”