Figure 3

Gold-labelled parC DNA binds as a ParR–parC complex to the ends of single ParM filaments. (A) Titration experiments to determine the optimal ParR and gold–streptavidin concentrations in relation to the amount of parC DNA used. Arrows indicate concentrations subsequently used. (B) Determination of the correct ParM concentration for the labelling experiment to minimise spontaneous filament formation in the absence of ParRC. (C–P) Electron micrographs showing ATP-polymerised ParM filaments with gold particles at the ends. ParR–parC complexes could also be observed binding along filaments (N) and complexes sometimes caused pairing of filaments (P). Scale bar 20 nm. (Q) Table quantifying binding events for ATP- and AMP-PNP-assembled filaments. Filaments were randomly imaged and classified as being gold-labelled at 0, 1 or both ends. The total number of side-binding events was counted and divided by the total number of filaments to give average number of complexes bound along the side per filament. (R) ADP-polymerised ParM (125 M) does not interact with ParRC complexes as detected by the biotin pull-down assay. Polymerisation was tested using the sedimentation assay.