Cleavage Assays

Cleavage assays can be particularly useful to determine if a potential drug targets gyrase. Some drugs interrupt the DNA breakage-reunion step of the gyrase reaction. This leads to cell death and it is the mechanism behind the action of the quinolones such as nalidixic acid and ciprofloxacin.

A typical reaction requires 10 times as much gyrase as a supercoiling reaction.

DNA gyrase is incubated with 0.3 µg of supercoiled pBR322 in a reaction volume of 30 µl at 37°C for 1 hour in 35 mM Tris.HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 6.5% (w/v) glycerol and 0.1 mg/ml albumin. 0.2% (w/v) SDS and 0.1 mg/ml Proteinase K are added before a further incubation at 37°C for 30 minutes.

The reaction is stopped by the addition of 3 µl 0.5 mM EDTA and 30 µl chloroform/iso-amyl alcohol (24/1) and 30 µl Stop Buffer (STEB), before being loaded on an agarose gel.

Gels can be run either in TBE (90 mM Tris.Borate, 2 mM EDTA) or TAE (40 mM Tris.acetate, 2 mM EDTA)

80-90% Cleavage can be obtained with 30 nM gyrase in a 30 µl reaction (see titration below).

cleavage diagram