Figure 3.

Presence of Topo IV does not increase the amount of MukB on the DNA. A, tailed, nicked DNA is an effective substrate for maximal DNA condensation. Reaction mixtures containing the tailed, nicked DNA, the indicated concentrations of MukB, and either in the presence (63 nm) or absence of Topo IV were incubated for 5 min at 37 °C, and the protein-DNA complexes formed were analyzed by gel electrophoresis as described in the legend to Fig. 1A. B, schematic of the pulldown experiment. TN DNA is tailed, nicked DNA C, pulldown assay to determine the amount of MukB on the DNA. Reaction mixtures contained the tailed, nicked DNA substrate bound to streptavidin-coated magnetic beads, MukB (63 nm), and Topo IV (63 nm) as indicated. After a 5-min incubation at 37 °C, the beads were pulled down on a magnet, and the supernatants (S) were removed. The pellet (P) was resuspended in SDS-PAGE loading buffer, and the supernatants were mixed 1:1 with 2× SDS-PAGE loading buffer. After heating at 100 °C for 5 min, identical volume equivalents of the reaction mixtures were subjected to SDS-PAGE. The proteins were transferred to membranes and Western-blotted with either anti-MukB or anti-ParC antibodies as described under “Experimental procedures.”