Fig. 3.
BfiI cleavage of immobilized DNA. (A) The experimental strategy. (B) A solution of 4 nM bio-30*/30 duplex (Table 1), whose top strand carries a biotin at its 5′ end and a 32P label at its 3′ end, was incubated with streptavidincoated magnetic beads in reaction buffer at pH 6.5 containing 50 nM BfiI for 5 min. The beads were washed twice with pH 6.5 buffer and then resuspended in pH 8.0 buffer. At timed intervals after the resuspension, the extent of cleavage of the top strand was monitored (○). In a parallel experiment, top-strand cleavage was measured under the same conditions except that the beads were resuspended in pH 8.0 buffer containing the K107A mutant of BfiI at 100 nM (•). The solid line is the optimal exponential fit to the latter data (rate constant 0.18 ± 0.02 min–1, amplitude 52%).