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.

Background: The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself. Following collision with a replication fork, the covalent complex can be converted to a double-strand break. Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished.

Integration host factor (IHF) is a small, basic protein that is needed for efficient recombination of bacteriophage lambda, as well as for other host and viral functions. We have constructed strains in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter. Separate overexpression of himA and hip led to the production of unstable and insoluble peptides, respectively. In contrast, the overexpression of both genes conjointly led to the accumulation of large amounts of active IHF. Extracts of such cells provided the starting material for a rapid purification procedure that results in milligram quantities of apparently homogeneous IHF.

Lambda's Int protein acts as a specific topoisomerase at attachment sites, the DNA segments that are required for site‐specific recombination. Int cleaves each strand of an attachment site at a unique place and creates strand exchanges by joining broken ends from two different parents. To study the action of Int topoisomerase in more detail, heteroduplex attachment sites were made by annealing strands that are complementary except for a few base pairs that lie in the region between the points of top and bottom strand exchange in the attachment site core. These heteroduplexes appear to interact normally with Int and its accessory proteins IHF and Xis. Although the heteroduplex sites are specifically cleaved by Int topoisomerase, rejoining of the broken DNA is hindered by the lack of Watson‐‐Crick complementarity adjacent to the break. Because of this, heteroduplexes accumulate broken intermediates which are then processed in novel ways. We have used this feature to provide new information about functional differences between attachment sites, to investigate the way Xis protein controls directionality of site‐specific recombination, and to demonstrate that Int protein can join strands indiscriminately and can therefore generate recombinants with either of two genetic polarities.