Analysis of Cleavage Complexes Using Reactive Inhibitor Derivatives

With recent progress in determining the crystal structures of type I and type II topoisomerases (topo I and topo II, respectively) (1 ,2 ), an understanding of the binding of DNA-cleavage-inducing inhibitors at the atomic level is within reach. A very useful approach for investigating the nature of the inhibitor binding site is the crosslinking of reactive inhibitor derivatives to the DNA or protein within the cleavage complex. In this chapter, we describe methods that have been used to crosslink a 4′-(9-acridinylamino)methanesulfon-m -anisidide (m -AMSA) derivative to the DNA within a topo II cleavage complex and a camptothecin (CPT) derivative to the DNA within a topo I cleavage complex. In both cases, the derivative reacted only with the base pairs immediately adjacent to the scissile phosphodiester bond and only in the presence of topoisomerase, providing strong and direct evidence that the DNA substrate forms an important component of the inhibitor binding site.