Difference Topology: Analysis of High-Order DNA-Protin Assemblies

DNA transactions in biological systems are often carried out by multisubunit protein assemblies that confer a defined topology on their DNA target sites. A subset of biochemically characterized site-specific recombination reactions and at least one DNA transposition reaction have been subject to extensive topological analysis. It is conceivable that interactions between the replication apparatus and origins of replication or those between transcription machineries and promoters, enhancers and repressor binding sequences also impose precise topological constraints on the path of DNA. Such ‘topological filters’ are thought to stabilize DNA-protein configurations that are conducive to triggering the chemical steps of the respective reactions. ‘Difference topology’ is a simple method for deciphering the DNA topology within complex DNA-protein machines that are not readily amenable to standard structural analyses. The logic is to trap the crossings formed by distinct DNA segments by tying them into knots or links by site-specific DNA inversion and deletion, respectively, carried out by a recombinase. The number of such crossings can then be counted by analytical methods such as gel electrophoresis or electron microscopy.