Relief of ParB autoinhibition by parS DNA catalysis and ParB recycling by CTP hydrolysis promote bacterial centromere assembly

Abstract

Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB protein acts as an adaptor between the 16 bp centromeric parS DNA sequences and the DNA segregation ATPase ParA. It accumulates at high concentrations at and near a parS site by assembling a partition complex. ParB dimers form a DNA sliding clamp whose closure at parS requires CTP binding. The mechanism underlying ParB loading and the role of CTP hydrolysis however remain unclear. We show that CTP hydrolysis is dispensable for Smc recruitment to parS sites in Bacillus subtilis but is essential for chromosome segregation by ParABS in the absence of Smc. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It recycles off-target ParB clamps to allow for new attempts at parS targeting and it limits the extent of spreading from parS by promoting DNA unloading. We also propose a model for how parS DNA catalyzes ParB clamp closure involving a steric clash between ParB protomers binding to opposing parS half sites.

ParS-independent recruitment of the bacterial chromosome-partitioning protein ParB

“…1E), during which time the ParB diffused away from the parS site. This residence time is in line with the previously determined CTP hydrolysis rate of 1 CTP per 100 s for ParBBSu (4), where the hydrolysis of CTP would cause the ParB clamp to destabilize and open, allowing dissociation from the DNA (4,5,19). Interestingly, we also observed a minor second population of molecules with roughly double the residence time of the first population (188 ± 18 s (average ± SEM), Fig.…”

“…S9A), which allows for the initial loading (clamp closing) but reduces opening of the ParB clamp upon hydrolysis (Fig. S9B-E) (4,7,19,25). This resulted in a low and length-independent passing fraction, indicating an essential role of CTP hydrolysis in the in-trans ParB-ParB recruitment (Fig.…”

“…Indeed, theoretical modelling of a ParB 'clamping and sliding' model indicated that such roadblocks dramatically limit the spreading distance on F-plasmids ( 28). Yet, in vivo ChIP-seq data do not show strong changes in the ParB occupancy in the vicinity of genes and operons (18)(19)(20)(21)(22)29).Here, we examine the mechanism of ParB spreading using a controlled DNA-stretching assay that allows in vitro visualization at the single-molecule level (30). First, we verified that CTP and the parS site are essential for the loading and diffusion of ParB on DNA.…”

“…A necessary feature for correct chromosome partitioning comes from a particular ability of ParB proteins to clamp the parS-DNA as a dimer (4,5) and to subsequently slide along the DNA by diffusion, effectively freeing the parS site for new ParB proteins to load. Indeed, ParB has been found to laterally spread over large genomic regions surrounding the parS sites in vivo (10-15 kb; (18)(19)(20)(21)(22)), which was reported to be essential for partition complex formation (9,10,16,18,19,(23)(24)(25). However, in vitro studies showed that single DNA-bound proteins block the diffusion of ParB along DNA very efficiently (4,7,17), raising the question how spreading can occur in a dense cellular environment, where, with ~1 gene and ~20-50 Nucleoid Associated Proteins (NAPs) per kb (26,27), sliding ParB dimers will continuously run into 'roadblocks' that will stall their movement.…”

“…Indeed, theoretical modelling of a ParB 'clamping and sliding' model indicated that such roadblocks dramatically limit the spreading distance on F-plasmids (28). Yet, in vivo ChIP-seq data do not show strong changes in the ParB occupancy in the vicinity of genes and operons (18)(19)(20)(21)(22)29).…”