The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.Topoisomerases are ubiquitous enzymes that play important roles in virtually all cells. The defining activity of these enzymes is their capacity to break DNA and, after an interval in which topological changes may occur, to reseal the break without the intervention of a high energy cofactor. Reversible breakage and reunion by topoisomerases is achieved through an intermediate in which a specific tyrosine residue of the enzyme is covalently joined to the DNA at the site of the break. The tyrosine-phosphodiester linkage is formed with one of two distinct polarities. Some enzymes join to the 5' end of the break, liberating a fragment terminated by a 3' hydroxyl; representatives include bacterial topoisomerase I as well as DNA gyrase and its relatives, the topoisomerase TI family. Other topoisomerase enzymes join to the 3' end of the break, liberating a fragment with a free 5' hydroxyl; representatives include the eukaryotic type I topoisomerases and the Int family of site-specific recombinases (for reviews, see refs. 1 and 2).The covalent intermediate between a topoisomerase and DNA is normally quite transient, i.e., strand rejoining is fast relative to strand cleavage. However, when a topoisomerase acts on DNA that contains imperfections (3, 4) or when specific inhibitors are present (5, 6), the rejoining step is slowed or blocked. This shifts the covalent complex from a transient intermediate toward a dead-end product; the associated long-lived break in the DNA backbone can have dire consequences for chromosome stability and cell survival (5,6). In this work, we describe an activity that hydrolyzes the covalent bond that joins topoisomerases to the 3' end of DNA. The specificity, robustness, and widespread distribution of this enzyme suggest that it could be an important element in the repair of DNA that has become stuck in covalent linkage with eukaryotic type I topoisomerases. MATERIALS AND METHODSDNA Substrates. Conventional oligonucleotides were constructed using an Applied Biosystems model 391 DNA synthesizer (Perkin-Elmer) or were purchased from Midland Certified Reagent (Midla...

Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death. Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described. Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair. The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized. The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.