DNA STRAND TRANSFER-REACTIONS CATALYZED BY VACCINIA TOPOISOMERASE - HYDROLYSIS AND GLYCEROLOLYSIS OF THE COVALENT PROTEIN-DNA INTERMEDIATE

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at si tes containing the sequence 5'-CCCTTdown arrow. The T-down arrow nucle otide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisom erase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor str and. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent interme diate and catalyze no detectable DNA hydrolysis. This suggests that hy drolysis occurs subsequent to formation of the covalent protein-DNA ad duct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is opt imal at pH 9.5.