Figure 1. Direct visualization of ParB specific binding to parS sites.

(A) Domains and functional motifs of ParB as reported previously (Bartosik et al., 2004; Kusiak et al., 2011). Mutations R80A, defective for cytidine triphosphate (CTP) binding, and R149G, defective for parS binding, are indicated. (B) ParB dimer cartoon showing dimerization through the central and C-terminal domains. The nucleotide binding site at the N-terminal domain (NTD) is also indicated. (C) Schematic representation of the single-length 39× parS DNA used for C-trap experiments. The DNA contains 39 parS sequences distributed in six groups forming two clusters separated by 1905 bp (39× parS DNA). The positions of the parS sites in the DNA cartoon are represented to scale. (D) Schematic of the C-trap experiment where single and tandem (double-length) tethers are immobilized between two beads and scanned with a confocal microscope using 488 nm illumination (upper part). Representative confocal images of the experiment under no CTP, 2 mM CTP, or 2 mM CTPγS conditions (lower part) and 20 nM ParB₂^AF488. Dark to bright regions correspond to a scale of 0–30 photon counts for single-length tethers and 0–50 counts for tandem tethers. (E) Representative profiles (500 nm width) of the fluorescence intensity along the DNA axis of the confocal images depicted in D (only single-length tether data). Positions of the parS sequences are included to scale in the background. Brighter regions between the beads correlate with the position of the parS clusters. ParB proteins are also observed outside the parS region (red arrow) and in general the fluorescence intensity outside the parS region is always above the background and larger in CTPγS compared to CTP experiments. (F) Quantification of fluorescence intensity at the parS-containing region under no CTP, CTP, and CTPγS conditions.

Figure 1—figure supplement 1. C-trap layout and nucleotide triphosphate (NTP) hydrolysis experiments.

(A) Cartoon of the fluid chamber employed for the experiments performed in this work. We used four laminar flows, containing streptavidin-coated beads, biotinylated DNA, buffer, or fluorescently labelled proteins. Confocal images and kymographs were obtained in channel 4 after a 2 min protein incubation. (B) NTP hydrolysis by wild-type ParB was measured using a colorimetric assay. Mean values are shown from four repeat experiments alongside the standard error of the mean. ParB hydrolyses cytidine triphosphate (CTP) in the absence of DNA at ~0.2 CTP molecules per dimer, per minute. CTP hydrolysis increases three- to four-fold in the presence of parS DNA. Nucleotides ATP, CTPγS, GTP, or UTP cannot be hydrolysed by ParB. The CTP hydrolysis rate increases linearly with the concentration of parS sequences. (C) NTP hydrolysis by wild-type ParB or ParB^AF488 was measured in the absence or presence of DNA. Mean values are shown from two repeat experiments alongside the standard error of the mean. ParB^AF488 retains parS-stimulated CTPase activity within two-fold levels of wild-type protein, which is likely attributable to inactivation of a fraction of the protein by the long labelling procedure.

Figure 1—figure supplement 2. Fabrication of small DNA plasmids.

Schematic representation of the steps followed to fabricate DNA plasmids containing multiple copies of parS which were used to prepare the magnetic tweezers DNA substrates.

Figure 1—figure supplement 3. Fabrication of large DNA plasmids.

Schematic representation of the steps followed to fabricate the large plasmids used to make the C-trap DNA substrates. This scheme specifically represents the cloning of the large 39× parS plasmid. Further details are described in the Supplementary methods section.

Figure 1—figure supplement 4. Kymographs of ParB bound along parS DNA.

(A) Fluorescence kymograph of a single 39× parS DNA molecule (Figure 1C) obtained with the C-trap including 20 nM ParB₂ and 2 mM cytidine triphosphate (CTP). A cartoon of the experiment with a schematic of the DNA showing the positions of the parS sequences plotted to scale is included on the left. (B) Same experiment as in A but using 2 mM CTPγS. Regions corresponding to the position of the parS groups could be clearly identified. (C) Average intensity profile (30 s) obtained along the DNA molecule. Positions of the parS sequences are included to scale in the background. The fluorescence intensity peaks correlate very well with the position of the six groups of parS sites. (D) Time evolution of the fluorescence intensity at the parS region obtained from kymographs under no CTP, CTP, cytidine diphosphate (CDP), and CTPγS conditions. Represented data are the average of multiple (no CTP: n=6, CTP: n=17, CTPγS: n=11, CDP: n=16) fluorescence intensity time courses. Dotted lines represent plus/minus the standard error of the mean. Time zero corresponds to the moment of turning on the excitation.