CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS

Abstract

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPgS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum-dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.

A CTP-dependent gating mechanism enables ParB spreading on DNA

“…We also noted that the ChIP-seq profile of CFP-ParB (E102A) in E. coli is highly asymmetrical, with more enrichment in the 2905–2911 kb region than the 2885–2899 kb region (shaded areas, Figure 8B ). The asymmetrical spreading is possibly due to an impediment in one direction by roadblocks such as RNA polymerases or DNA-bound proteins, which have been shown previously to be able to interfere with ParB spreading ( Balaguer F de et al, 2021 ; Breier and Grossman, 2007 ; Jalal et al, 2020c ; Murray et al, 2006 ; Rodionov et al, 1999 ; Soh et al, 2019 ).…”

“…The ParB-DNA nucleoprotein complex stimulates the ATPase activity of ParA, driving the movement of the parS locus (and subsequently, the whole chromosome) to the opposite pole of the cell ( Hwang et al, 2013 ; Leonard et al, 2005 ; Lim et al, 2014 ; Taylor et al, 2021 ; Vecchiarelli et al, 2014 ; Vecchiarelli et al, 2012 ). ParB spreads by sliding along the DNA, in a manner that depends on the binding of a co-factor, cytidine triphosphate (CTP) ( Balaguer F de et al, 2021 ; Jalal et al, 2020c ; Osorio-Valeriano et al, 2019 ; Soh et al, 2019 ). A co-crystal structure of a C-terminal domain truncated Bacillus subtilis ParB (ParB∆CTD) together with CDP showed the nucleotide to be sandwiched between adjacent subunits, thus promoting their dimerization ( Soh et al, 2019 ).…”

Recent Advances of Optical Tweezers–Based Dynamic Force Spectroscopy and Mechanical Measurement Assays for Live-Cell Mechanobiology

“…For example, by combining holographic OT with a spinning disk confocal fluorescence microscope, Wolfson et al enabled simultaneously manipulating on multiple objects while continuously characterizing their intracellular signaling events with high spatiotemporal resolution [80]. Meanwhile, Balaguer et al used a similar system to be the first to visualize the direct interaction between ParB proteins and the parS loading site [81]. Additionally, the combination of OT with FRET-based molecular force microscopy also enables the concurrent quantification of force experienced by a cell and signal mechanotransduction within the cell [82].…”

The CTP-binding domain is disengaged from the DNA-binding domain in a co-crystal structure of Bacillus subtilis Noc-DNA complex

“…In B. subtilis, noc resulted from parB via a gene duplication and neo-functionalization event 10,16 , and both Noc and ParB are CTPdependent molecular switches 7,8,[17][18][19][20] . CTPbinding switches nucleating ParB/Noc (bound at a high-affinity parS/NBS site) from an openclamp conformation (Figure 5A-B) to a closedclamp conformation that can escape from parS/NBS to slide to neighboring DNA while still entrapping DNA (Figure 5C) 6,7,14,17,18 . The closed-clamp conformation is possible due to the new dimerization interface between the two adjacent N-terminal CTP-binding domains of ParB/Noc (the so-called NTD-NTD engagement, Figure 5C) 6,7,14,17 .…”

“…In B. subtilis, noc resulted from parB via a gene duplication and neo-functionalization event 10,16 , and both Noc and ParB are CTP-dependent molecular switches 7,8,17–20 . CTP-binding switches nucleating ParB/Noc (bound at a high-affinity parS/NBS site) from an openclamp conformation (Figure 5A-B) to a closed-clamp conformation that can escape from parS/NBS to slide to neighboring DNA while still entrapping DNA (Figure 5C) 6,7,14,17,18 . The closed-clamp conformation is possible due to the new dimerization interface between the two adjacent N-terminal CTP-binding domains of ParB/Noc (the so-called NTD-NTD engagement, Figure 5C) 6,7,14,17 .…”