REPLACEMENT OF THE ACTIVE-SITE TYROSINE OF VACCINIA DNA TOPOISOMERASEBY GLUTAMATE, CYSTEINE OR HISTIDINE CONVERTS THE ENZYME INTO A SITE-SPECIFIC ENDONUCLEASE

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5' -CCCTT down arrow sites in duplex DNA, The T down arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidin e in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp dupl ex DNA at the CCCTT down arrow site to yield a 3' phosphate-terminated product, The Cys-274 mutant forms trace levels of a covalent protein- DNA complex, suggesting that the DNA cleavage reaction may proceed thr ough a cysteinyl-phosphate intermediate, However, the His-274 and Glu- 274 mutants evince no detectable accumulation of a covalent protein-DN A adduct. Glu-274 is the most active of the mutants tested, The pH dep endence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covale nt adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reac tion is slower by 5-6 orders of magnitude than the rate of covalent ad duct formation by the wild-type topoisomerase, but is similar to 20 ti mes faster than the rate of hydrolysis by the wild-type covalent adduc t, We discuss two potential mechanisms to account for the apparent con version of a topoisomerase into an endonuclease.