ParB spreading requires DNA bridging

Abstract

The parABS system is a widely employed mechanism for plasmid partitioning and chromosome segregation in bacteria. ParB binds to parS sites on plasmids and chromosomes and associates with broad regions of adjacent DNA, a phenomenon known as spreading. Although essential for ParB function, the mechanism of spreading remains poorly understood. Using single-molecule approaches, we discovered that Bacillus subtilis ParB (Spo0J) is able to trap DNA loops. Point mutants in Spo0J that disrupt DNA bridging are defective in spreading and recruitment of structural maintenance of chromosomes (SMC) condensin complexes in vivo. DNA bridging helps to explain how a limited number of Spo0J molecules per parS site (~20) can spread over many kilobases and suggests a mechanism by which ParB proteins could facilitate the loading of SMC complexes. We show that DNA bridging is a property of diverse ParB homologs, suggesting broad evolutionary conservation.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

“…14), the minimal density of ParB is ∼1,000/μm 2 , consistent with estimates of ParB density on P1 plasmid of approximately one dimer per 30 bp (25). This points to the critical role of the "ParB-spreading" process in which ParB binding to parS on the plasmid nucleates higher-order complex assembly involving a large number of ParBs to promote plasmid segregation (26)(27)(28)(29)(30). Conversely, if the density of ParB was too high, too many ParA-ParB bonds formed resulting in the decrease of both the persistence and the speed (Fig.…”

Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis

“…To determine whether the ParB/parS nucleoprotein complexes or condensin itself was required for the long-range interactions, we used a B. subtilis strain harboring Vibrio cholerae ParB1 [referred to as ParB1(Vc)] that forms nucleoprotein complexes at parS sites but is unable to recruit condensin. ParB1(Vc) recognizes the identical parS site in vivo (Livny et al 2007), and single-molecule experiments in vitro indicate that ParB1(Vc) mediates nearest neighbor and bridging interactions similar to B. subtilis ParB (Graham et al 2014). Figure 2B shows that B. subtilis cells harboring an mCherry-ParB1(Vc) fusion in a strain lacking the endogenous parB gene generated fluorescent foci that depend on the origin-proximal parS sites.…”

“…To determine whether these nucleoprotein complexes are required for long-range interactions, we performed Hi-C on cells harboring a ParB point mutant (G77S) that binds parS sites with affinity similar to wild type but does not spread to neighboring sites (Breier and Grossman 2007;Graham et al 2014). The interaction map for this mutant resembled those derived from the ΔparB and parSΔ9 strains ( Fig.…”

“…All 10 sites are occupied by ParB in vivo as assayed by ChIP, although the origin-distal sites (especially the one at −161°) appear to be weaker than those present near the origin (Supplemental Fig. S5; Breier and Grossman 2007;Graham et al 2014). Using a chromosome organization assay that takes advantage of polar division Representative fluorescence images of sporulating wild-type (BWX941) and ΔparAB (BWX3327) cells harboring an ScpB-YFP fusion.…”

“…ParB proteins form large nucleoprotein complexes at parS sites that span 10-20 kb of flanking DNA (Murray et al 2006;Breier and Grossman 2007) mediated by nearest neighbor and bridging interactions (Graham et al 2014;Taylor et al 2015). To determine whether these nucleoprotein complexes are required for long-range interactions, we performed Hi-C on cells harboring a ParB point mutant (G77S) that binds parS sites with affinity similar to wild type but does not spread to neighboring sites (Breier and Grossman 2007;Graham et al 2014).…”