J. Mol. Biol. (1996) 259, 317–324 COMMUNICATION Topoisomerase II-mediated DNA Cleavage: Evidence for Distinct Regions of Enzyme-DNA Contacts Jan Alsner, Henrik V. So rensen, Vibe K. Schmidt, Boe S. So rensen and Ole Westergaard* To determine the specific interaction sites of topoisomerase II within the Department of Molecular and Structural Biology DNA region defined by the footprint of the enzyme, we have investigated the cleavage reaction on double-stranded DNA substrates containing nicks University of Aarhus, C.F. Mo  llers Alle ´, DK-8000 Århus and deletions. Topoisomerase II-mediated cleavage of the DNA substrates is suicidal as the enzyme is unable to religate the cleaved DNA due to C, Denmark diffusion of the small nucleotides 5' to the cleavage position. Thus, suicidal cleavage is obtained with substrates having one, two or three nucleotides 5' to the cleavage position. The enzyme requires interaction with three distinct regions of double-stranded DNA for cleavage to occur, one region spanning the eight nucleotides located around the cleavage position and two distal regions each spanning approximately six nucleotides. A model is proposed, where these data are taken to imply that two distinct regions of interactions exist between each topoisomerase II subunit and its DNA substrate. The model is discussed in relation to the recently solved three-dimensional structure of yeast topoisomerase II.  1996 Academic Press Limited Keywords: topoisomerase II; mammalian; suicide DNA substrate; *Corresponding author cleavage/religation reaction; DNA interaction domains Eukaryotic DNA topoisomerase II is a highly conserved, ubiquitous enzyme that modulates the topological state of DNA. The enzyme is required for segregation of mitotic chromosomes (Uemura et al ., 1987), plays a critical role in chromosome condensation (Wood & Earnshaw, 1990; Adachi et al ., 1991; Swedlow et al ., 1993), and is involved in maintenance of mitotic chromosomes (Earnshaw et al ., 1985; Earnshaw & Heck, 1985; Gasser et al ., 1986; Cockerill & Garrard, 1986). Topoisomerase II is also involved in the processes of DNA replication, transcription and recombination (reviewed by Hsieh, 1990; Sternglanz, 1989). Topoisomerase II catalyzes the interconversion of DNA topoisomers by cleaving both strands of a DNA double helix, passing another DNA duplex through the break, and religating the broken strands (reviewed by Vosberg, 1985; Osheroff et al ., 1991). During the transient double-strand breakage the enzyme is covalently attached to the 5' termini of the DNA. A number of potent antineoplastic agents stabilize the covalent topoisomerase II-DNA inter- mediate (Osheroff, 1989a; Robinson & Osheroff, 1990; So rensen et al ., 1992), which is most likely the basis of their antitumor activity (reviewed by Liu, 1989). Various forms of DNA molecules are substrates for topoisomerase II. On double-stranded DNA, topoisomerase II binds supercoiled plasmid DNA more efficiently than relaxed or linear DNA (Osheroff, 1986; Roca & Wang, 1992). With respect to DNA cleavage, the majority of naturally occurring sites described so far are recognized on double-stranded DNA (Wang,1985; Osheroff, 1989b, 1991). Based on sequences that are cleaved when localized in double-stranded DNA (Sander & Hsieh, 1983), sites have been created that contain a double-stranded/single-stranded junction (Lund et al ., 1990; Andersen et al ., 1991). DNA cleavage is Present addresses: J. Alsner, Department of Experimental Clinical Oncology, The Danish Cancer Society, No rrebrogade, DK-8000 Århus C, Denmark; H.V. So rensen, Nuffield Department of Clinical Medicine, John Ratcliffe Hospital, Oxford OX3 9DU, UK; B.S. So rensen, Department of Clinical Biochemistry, Aarhus University Hospital, No rrebrogade, DK-8000 Århus C, Denmark. Abbreviations used: m-AMSA or amsacrine, 4'-(9-acridinylamino)methanesulphon-m-anisidide; VM-26 or teniposide, 4'-demethylepipodophyllotoxin thenylidene--D-glucoside. 0022–2836/96/230317–08 $18.00/0  1996 Academic Press Limited