Figure 6.

Substitution of −1 guanines at the pBR322 ‘1073 site’ with adenine, cytosine or thymine abolishes gyrase DNA cleavage stimulated by CL but not by gemifloxacin. The 295-bp pBR322 fragment 5′ ³³P-labelled on the bottom strand was amplified by PCR from pBR322 plasmids carrying the wild-type site (G at both −1 positions, indicated by WT) or mutant sites bearing A, C or T at both −1 positions (denoted by −1A, −1C and −1T). The fragments were used in a DNA cleavage assay with gyrase and 1 mM ATP and either 100 μM gemifloxacin (lanes 1) or 200 μM CL (lanes 2 and 3). DNA was recovered by ethanol precipitation and run on an 6% denaturing polyacrylamide gel alongside ACGT chain termination DNA sequencing products obtained from a wild-type DNA template and G+A Maxam–Gilbert chemical sequencing products generated for each of the four substrates. Prior to electrophoresis, one set of CL cleavage products was treated with hot piperidine (lanes 3). Open circle denotes cleavage product derived from the major cleavage site.