A topological analysis of difference topology experiments of condensin with Topoisomerases II

ABSTRACTAn experimental technique called difference topology combined with the mathematics of tangle analysis has been used to unveil the structure of DNA bound by the Mu transpososome. However, difference topology experiments can be difficult and time-consuming. We discuss a modification that greatly simplifies this experimental technique. This simple experiment involves using a topoisomerase to trap DNA crossings bound by a protein complex and then running a gel to determine the crossing number of the knotted product(s). We develop the mathematics needed to analyze the results and apply these results to model the topology of DNA bound by 13S condensin and by the condensin MukB.SUMMARY STATEMENTTangles are used to model protein-DNA complexes: A 3-dimensional ball represents protein while strings embedded in this ball represent protein-bound DNA. We use this simple model to analyze experimental results.

Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.

Determining the topology of stable protein–DNA complexes

Difference topology is an experimental technique that can be used to unveil the topological structure adopted by two or more DNA segments in a stable protein–DNA complex. Difference topology has also been used to detect intermediates in a reaction pathway and to investigate the role of DNA supercoiling. In the present article, we review difference topology as applied to the Mu transpososome. The tools discussed can be applied to any stable nucleoprotein complex.