Topo IV Relaxation Assays

A typical reaction is as follows:

1 U of topo IV is incubated with 0.4 µg supercoiled pBR322 DNA in a 30 µl reaction at 37°C for 30 minutes under the following conditions: 40 mM HEPES.KOH (pH 7.6), 100 mM Potassium Glutamate, 10 mM Magnesium Acetate, 10 mM DTT, 1 mM ATP, and 50 µg/ml albumin.

The reaction is stopped by the addition of 30 µl chloroform/iso-amyl alcohol (24/1) and 30 µl 2X 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) although the resolution of supercoiled pBR322 is much better if run in TAE.

Explanation of Gels

diagram relaxation

The substrate used usually consists of two bands on a gel. The upper one is open-circular (nicked) DNA and the faster migrating band is negatively supercoiled (closed circular) plasmid.

Treatment of negatively supercoiled pBR322 with topo IV converts the supercoiled form to the relaxed form and this consists of a series of bands on a gel which are different topoisomers (DNAs of different linking number).

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 DNA. If this occurs, change your buffers including the water. If this does not resolve the problem it is probably due to contamination of the enzyme/cell extract itself.