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. 2020 Feb 20;9:e53515. doi: 10.7554/eLife.53515

Figure 1. CTP reduces the nucleation of Caulobacter ParB at parS.

(A) The domain architecture of ParB (dark green) and TetR (grey), and their respective DNA-binding sites parS and tetO. Convergent arrows below DNA-binding sites indicate that parS and tetO are palindromic. (B) Bio-layer interferometric (BLI) analysis of the interaction between a premix of 1 μM ParB-His6 dimer ± 1 mM NTP and a 20 bp DNA duplex containing parS. Biotinylated DNA fragments were immobilized onto the surface of a Streptavidin (SA)-coated probe (See Materials and methods). The BLI probe was dipped into a buffer only solution (0–30 s), then to a premix of protein ± NTP (30–150 s: association phase), and finally returned to a buffer only solution (150–270 s: dissociation phase). Sensorgrams were recorded over time. BLI analysis of the interaction between 1 μM TetR-His6 and a 20 bp parS probe was also recorded (a negative control). (C) BLI analysis of the interaction between a premix of 1 μM TetR-His6 ± 1 mM NTP and a 28 bp DNA duplex containing tetO. BLI analysis of the interaction between 1 mM CTP and a 28 bp tetO probe was also recorded (a negative control). (D) BLI analysis of the interaction between 1 μM Caulobacter ParB-His6 (without CTP) and a 20 bp parS DNA. For the dissociation phase, the probe was returned to a buffer only or buffer supplemented with 1 mM CTP. All buffers used for experiments in this figure contained Mg2+. Each BLI experiment was triplicated and a representative sensorgram was presented.

Figure 1—source data 1. Data used to generate Figure 1.

Figure 1.

Figure 1—figure supplement 1. ParB and TetR bind specifically to their cognate-binding sites parS and tetO, respectively.

Figure 1—figure supplement 1.

(A) BLI analysis of the interaction between purified Caulobacter ParB-His6 (0.125 to 2 µM) and a 20 bp parS DNA probe or a 28 bp tetO DNA probe. (B) BLI analysis of the interaction between purified TetR-His6 (0.125 to 2 µM) and a 20 bp parS DNA probe or a 28 bp tetO DNA probe.
Figure 1—figure supplement 1—source data 1. Data used to generate Figure 1—figure supplement 1.
Figure 1—figure supplement 2. BLI analysis of the interaction between purified Caulobacter ParB and NTP in buffers lacking Mg2+.

Figure 1—figure supplement 2.

Caulobacter ParB-His6 (1 µM dimer)±1 mM NTP, and a 20 bp DNA duplex containing parS were used. Mg2+ was omitted from all buffers in this experiment. A schematic of the DNA substrate is shown above the sensorgram.
Figure 1—figure supplement 2—source data 1. Data used to generate Figure 1—figure supplement 2.
Figure 1—figure supplement 3. BLI analysis of the interaction between purified Caulobacter ParB and cytidine mono-, di-, or triphosphate.

Figure 1—figure supplement 3.

(A) BLI analysis of the interaction between purified Caulobacter ParB and an increasing concentration of CTP-Mg2+. ParB-His6 (1 µM dimer) + 0–10 mM CTP, and a 20 bp DNA duplex containing parS were used for this experiment. (B) BLI analysis of the interaction between a premix of 1 μM Caulobacter ParB-His6 ± 1 mM cytidine mono-, di-, or triphosphate, and a 20 bp parS DNA. A schematic of the DNA substrate is shown above the sensorgram. Mg2+ was included in all buffers for experiments in this figure.
Figure 1—figure supplement 3—source data 1. Data used to generate Figure 1—figure supplement 3.