Figure 4.

Specific looping of SfiI confirmed with the TPM assay. Histograms of the RMS motion with increasing protein concentration reveal specific looping. Both panels show the data of a single tether and each histogram contains at least 15 × 10⁶ counts. The left panel shows the behaviour of the WT SfiI enzyme on the single site substrate in a buffer containing Ca²⁺. The tether remains unlooped with increasing WT concentrations up to 1 nM. At 10 nM enzyme, the non-specific interactions of multiple SfiI molecules condense the tether. The right panel shows the equilibrium between looped and unlooped state with increasing D79A concentration in Mg²⁺ buffer. At 10 nM, D79A also condenses the DNA, similar to the WT enzyme, indicating that the non-specific interactions are not altered by the mutation.