Figure 5.

Modes of action by AlfI and CspCI. The reaction in (a) contained, in 200 μl AloI buffer with 50 μM AdoMet (Table 2) at 20 °C, AlfI endonuclease (2 U) and 5 nM pIS2 (a plasmid with two AlfI sites). The reaction in (b) contained, in 200 μl of BaeI buffer (Table 2) at 37 °C, CspCI (2 U) and 5 nM pIS1 (a plasmid with two CspCI sites). In both reactions, the substrate was ³H-labelled and ∼85% was initially in its supercoiled state. Samples were taken from the reactions at the times indicated and analysed as described in Materials and Methods to determine concentrations of the following forms of the DNA: the supercoiled plasmid (marked SC, in red), the nicked form (OC, in blue), the full-length linear form with at least one double-strand break at one recognition site (FLL, in green) and the mean of the two linear products with double-strand breaks at both sites (L1/L2, in purple).