A NOVEL SUICIDE SUBSTRATE FOR DNA TOPOISOMERASES AND SITE-SPECIFIC RECOMBINASES

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 ph osphorothioate linkage creates an efficient suicide substrate for calf thymus topoisomerase I and lambda integrase protein (Int). Although t he bridging phosphorothioate linkage is cleaved by these enzymes, the 5'-sulfhydryl which is generated is not competent for subsequent ligat ion reactions. We use the irreversibility of Int-promoted cleavage to explore conditions and factors that contribute to various steps of lam bda integrative recombination, The phosphorothioate substrates offer a dvantages over conventional suicide substrates, may be potent tools fo r inhibition of the relevant cellular enzymes and represent a unique t ool for the study of many other phosphoryl transfer reactions.