Topoisomerase II-mediated DNA cleavage: evidence for distinct regions of enzyme-DNA contacts.

To determine the specific interaction sites of topoisomerase II within the DNA region defined by the footprint of the enzyme, we have investigated the cleavage reaction on double-stranded DNA substrates containing nicks and deletions. Topoisomerase II-mediated cleavage of the DNA substrates is suicidal as the enzyme is unable to religate the cleaved DNA due to 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.

Intramolecular and intermolecular DNA ligation mediated by topoisomerase II.

The ability of topoisomerase II to mediate a number of DNA rearrangements was examined at the molecular level. For this purpose a new type of defined donor and acceptor substrate have been developed, and used for studies of the intramolecular and intermolecular DNA ligation reactions of topoisomerase II. Intramolecular ligation occurred only to single-stranded acceptor molecules with the ability to base-pair to the donor substrate, while the intermolecular ligation reaction displayed a strong preference for double-stranded acceptor molecules with a short four base, single-stranded region. The efficiency of the intermolecular ligation reaction was highly dependent on base-pairing between the acceptor molecule and the DNA donor cleaved by topoisomerase II. Thus, acceptor molecules containing a blunt end or a four base 5' overhang without base-pairing ability ligated with an approximately eightfold reduced efficiency, as compared with the base-pairing control. Experiments demonstrated that the enzyme can ligate DNA molecules, where nucleotides were either removed or inserted in the employed acceptor molecules. The results indicate that topoisomerase II might be responsible for similar rearrangements in vivo, since gapped and nicked DNA structures appear as intermediates in processes such as replication and repair. The reaction is, however, likely to be constrained by the requirement of base-pairing for ligation.