A novel suicide substrate for DNA topoisomerases and site-specific recombinases

Abstract

DNA topoisomerases and DNA site-specific recombinases are biologically important enzymes involved in a diverse set of cellular processes. We show that replacement of a phosphodiester linkage by a 5'-bridging phosphorothioate linkage creates an efficient suicide substrate for calf thymus topoisomerase I and lambda integrase protein (Int). Although the bridging phosphorothioate linkage is cleaved by these enzymes, the 5'-sulfhydryl which is generated is not competent for subsequent ligation reactions. We use the irreversibility of Int-promoted cleavage to explore conditions and factors that contribute to various steps of lambda integrative recombination. The phosphorothioate substrates offer advantages over conventional suicide substrates, may be potent tools for inhibition of the relevant cellular enzymes and represent a unique tool for the study of many other phosphoryl transfer reactions.