The CTPase activity of ParB acts as a timing mechanism to control the dynamics and function of prokaryotic DNA partition complexes

Abstract

SUMMARYDNA partitioning CTPases of the ParB family mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromeric region of target DNA molecules and then spread laterally to form large nucleoprotein complexes that serve as docking points for the DNA segregation machinery. Here, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. Moreover, we solve crystal structures of ParB in the pre- and post-hydrolysis state and provide insights into the catalytic mechanism underlying nucleotide hydrolysis. The characterization of CTPase-deficient ParB variants reveals that CTP hydrolysis serves as a timing mechanism to control the sliding time of ParB. Hyperstable clamps are trapped on the DNA, leading to excessing spreading and severe chromosome segregation defects in vivo. These findings clarify the role of the ParB CTPase cycle in partition complex dynamics and function and thus complete our understanding of this prototypic CTP-dependent molecular switch.

ParB proteins can bypass DNA-bound roadblocks via dimer-dimer recruitment

“…S3, B and C; and table S2). The delayed dissociation may originate from nonspecific interaction of the open ParB clamp with the DNA via its positive residue patch at the C terminus ( 31 , 33 ).…”

“…In trans recruitment, in particular, would involve a transient loop formation between two distal segments on the DNA molecule. It has been previously proposed that ParB-like proteins can spread via a similar in trans mechanism ( 38 ), whereas previous work on ParB indicated that long loops may underlie ParB-DNA condensation ( 39 ), particularly at very low forces exerted in magnetic tweezer experiments ( 7 , 31 , 33 ) or DNA curtains ( 10 , 13 ). In our case, a loop would form transiently, and the ParB-ParB dimer-dimer connection would break following the recruitment event.…”

In vivo Assembly of Bacterial Partition Condensates on Circular Supercoiled and Linear DNA

“…The predominant fraction of intracellular ParB dimers (> 90%) forms densely packed clusters assembled at parS sites (Sanchez et al, 2015). These ParB condensates exhibit dynamic characteristics, with a high exchange of ParB rate between condensates (Debaugny et al, 2018;Osorio-Valeriano et al, 2021;Tisma et al, 2023). Upon binding to parS and CTP, ParB dimers undergo an important conformational change, transitioning from parS binding to clamping onto flanking DNA (see Figure 1B).…”

“…This latter was proposed based on the finding that ParB is a CTPase that clamps over parS DNA and subsequently diffuses along the DNA until the clamp opens (Figure 1B). The physical modeling of ParB DNA profiles through an exponential decay of ParB binding probability along the parS-proximal DNA could depict the 'C&S' on naked DNA (Osorio-Valeriano et al, 2021;Walter et al, 2020). However, this model did not align with some biochemical characteristics of ParB, especially the rates of release from parS and of unloading from the DNA (Walter et al, 2020).…”

“…Interestingly, it has also been shown that partition complexes are dynamic structures that display some properties of a liquid-droplet, i.e., an assembly that may be mediated by phase separation (Azaldegui et al, 2020). These properties include (i) the rapid exchange of ParB between separate partition complexes in the order of a few minutes (Debaugny et al, 2018;Osorio-Valeriano et al, 2021), (ii) the intracellular mobility of ParB is ~100 times slower inside ParB clusters (concentrated phase) than outside (diluted phase) (Guilhas et al, 2020), (iii) the ability of ParB foci to fuse when ParA is degradated (Guilhas et al, 2020), and (iv) the self-organization of ParB into droplets in vitro (Babl et al, 2022). Which mechanisms are at play to assemble these high molecular weight structures, essential for DNA segregation, is not yet fully understood.…”