Figure 7.

DNA binding. (a and b) The mixtures contained, in 10 µl buffer R*C, 20 nM DNA, either HEX-42S (a) or HEX-42NS (b), and BcgI at one of the following levels, from left to right: 0, 20, 40, 60, 80, 100, 150 or 200 nM. After 5 min at 37°C, the samples were subjected to electrophoresis through polyacrylamide and the fluorescence from each band recorded. On the left of gel images, F marks the position of the free DNA; C, the DNA–protein complexes that entered the gel; and W, the bottom of the wells. (c) From titrations of both HEX-42S and HEX-42NS with increasing amounts of BcgI protein, and from additional experiments with 24 bp duplexes (HEX-24S and HEX-24NS: gels not shown), the amount of free DNA in each sample was determined relative to the amount in a parallel lane without enzyme. The % DNA in the free form is shown as a function of the BcgI concentration: HEX-42S, white triangle; HEX-42NS, white squares; HEX-24S, black triangles; HEX-24NS, black squares. Mean values from three repeats are given: error bars show standard deviations. The red lines through the data with the specific duplexes denote stoichiometric binding where all of the added BcgI is bound to DNA until saturation of the DNA is achieved.