PMC

MukB and ParC interact in vitro and in vivo. (A) Either HA-ParC protein (86 kDa) or HA-GyrA protein (99 kDa) conjugated to anti-HA antibody agarose beads were incubated with MukB (170 kDa) in binding assays as described. The reaction was analyzed by SDS-PAGE. The 55-kDa band on the gel is the IgG heavy chain. (B Left) Monoclonal anti-HA antibody was used to immunoprecipitate MukB-HA₃ (174 kDa) from crude extracts prepared from BW30270mukB-HA₃ cells, and the gel was Western blotted using polyclonal anti-ParC antibody. (B Right) Polyclonal anti-ParC antibody was used to immunoprecipitate ParC from crude extract prepared from BW30270mukB-HA₃ cells, and the gel was Western blotted using monoclonal anti-HA antibody.

MukB interacts with the CTD of ParC. (A) Schematic of ParC and the designed deletion mutants. ParC protein consists of an NTD (green) and CTD (blue), which are connected by a flexible linker region (yellow). The CTD consists of five structurally similar units of a β-pinwheel (), the blades, which are denoted as B1, B2, B3, B4, and B5, ordered from the N terminus to the C terminus. The Δ696 mutant lacks the most C-terminal blade, B5. (B) The ParC HA-NTD (59 kDa) and the HA-ParCΔ696 (79 kDa) variant that lacks the fifth blade of the CTD do not interact with MukB. (C) ParC HA-CTD (29 kDa) is sufficient to interact with MukB. (D) Neither HA-ParC R705E, HA-ParC R729A, nor HA-ParC R705E R729A interact with MukB. Binding experiments were as described in the legend to .

Topo IV reconstituted with ParE and ParC R705E R729A is active. (A) ParC R705E R729A is as active as wild-type ParC in the unlinking of DNA rings. Assays for the decatenation of kinetoplast DNA by the indicated concentrations of Topo IV reconstituted with ParE and either wild-type or ParC R705E R729A were as described. MC, minicircle; CI, catenated intermediates. (B) The amount of minicircles released as shown in A was determined, and the means from three independent experiments were plotted. SEMs are indicated. (C) ParC R705E R729A is as active as wild-type ParC in the removal of negative supercoils. Assays for the relaxation of negatively supercoiled plasmid DNA by the indicated concentrations of Topo IV reconstituted with ParE and either wild-type ParC or ParC R705E R729A were as described. (D) The fraction of relaxed plasmid DNA as shown in C was determined, and the means from three independent experiments were plotted. SEMs are indicated. FI, form I DNA (supercoiled); FI′, form I′ DNA (covalently closed, relaxed); FII, form II DNA (nicked circular). A small amount of residual FII DNA present in the preparation of form I DNA comigrates with FI′ DNA.

Effect of MukB on Topo IV activity. (A and B) Effect of MukB on the decatenation activity of Topo IV (0.125 nM) reconstituted with either wild-type ParC (A) or ParC R705E R729A (B). (C) The extent of stimulation of decatenation by Topo IV in A and B was determined, and the means from three independent experiments were plotted. SEMs are indicated. (D) MukB stimulates the superhelical DNA relaxation activity of wild-type Topo IV (6 nM). (E) Topo IV (6 nM) reconstituted with ParE and ParC R705E R729A is unaffected by MukB. (F) The fraction of relaxed plasmid DNA in D and E was determined, and the means from three independent experiments were plotted. SEMs are indicated. (G) MukB does not affect the supercoiling activity of DNA gyrase (0.5 nM). Reaction mixtures containing form I′ DNA and the indicated concentrations of DNA gyrase and MukB were incubated and analyzed as described. (H) The fraction of supercoiled plasmid DNA in G was determined, and the average of two independent experiments was plotted.