Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3 '-para-nitrophenyl phosphate ester

DNA topoisomerases and DNA site-specific recombinases are involved in a div erse set of cellular processes but both function by making transient breaks in DNA, Type IB topoisomerases and tyrosine recombinases cleave DNA by tra nsesterification of an active site tyrosine to generate a DNA-3'-phosphotyr osyl-enzyme adduct and a free 5'-hydroxyl (5'-OH). Strand ligation results when the 5'-OH attacks the covalent complex and displaces the enzyme. We de scribe the synthesis of 3'-phospho-(para-nitrophenyl) oligonucleotides (3'- pNP DNAs), which mimic the natural 3'-phosphotyrosyl intermediate, and demo nstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase, Ligation occurs by direct attac k of a 5'-OH strand on the 3'-pNP DNA (i.e., without a covalent protein-DNA intermediate) and generates free para-nitrophenol as a product. The chromo genic DNA substrate allows ligation to be studied in real-time and in the a bsence of competing cleavage reactions and can be exploited for high-throug hput screening of topoisomerase/recombinase inhibitors.