Gyrase Supercoiling

A typical reaction is as follows:

1 U of DNA gyrase is incubated with 0.5 µg of relaxed pBR322 DNA in a 30 µl reaction at 37°C for 30 minutes under the following conditions: 35 mM Tris.HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM Spermidine, 1 mM ATP, 6.5% (w/v) glycerol and 0.1 mg/ml BSA.

Each reaction is stopped by the addition of 30 µl chloroform/iso-amyl alcohol (24/1) and 30 µl Stop Buffer (STEB:  40% sucrose, 100 mM Tris.HCl ( pH 7.5), 1 mM EDTA, 0.5 mg/ml bromophenol blue), before being loaded on an agarose gel. (0.8%-1.0%: w/v)

Gels can be run either in TBE (90 mM Tris.Borate, 2 mM EDTA) or TAE (40 mM Tris.acetate, 2 mM EDTA) although the resolution of supercoiled pBR322 is much better if run in TAE.

Explanation of Gels

diagram supercoiling

The substrate used (in this case relaxed pBR322) usually consists of a series of bands on a gel which are topoisomers (DNAs of different linking number).

Treatment of relaxed pBR322 with DNA gyrase converts the relaxed topoisomers to the supercoiled form of the plasmid which migrates faster on an agarose gel. An upper band may also be visible which is open-circular (nicked) DNA which is present in the relaxed substrate but co-migrates with some of the relexed topoisomers.

Intercalators

Problems can arise in assays if there are contaminants such as ethidium bromide or Chloroquine in the gel tanks. These chemicals are intercalators and can affect the mobility of the DNA leading to confusing results. If there is an intercalator present in the buffer the relaxed topoisomers have increased mobility and run in roughly the same place as negatively supercoiled plasmid. Depending on the amount of intercalator, the negatively supercoiled DNA may have slightly less mobility than normal.

Nucleases

If there are contaminating nucleases present in the assay (due to contaminated assay buffers or impure fractions from cell expression) there may be a significant increase in nicking leading to an increase in the amount of open-circular DNA and possibly the production of linear DNA. This runs as a single band between the OC and SC.

ATP

A loss of activity with previously active enzyme may be due to deterioration of the ATP in the buffer although the 5X buffer has been developed to minimize this. If loss of activity occurs, the addition of extra ATP to the reaction will overcome the problem.