INTRAMOLECULAR AND INTERMOLECULAR DNA LIGATION-MEDIATED BY TOPOISOMERASE-II

The ability of topoisomerase II to mediate a number of DNA rearrangeme nts was examined at the molecular level. For this purpose a new type o f defined donor and acceptor substrate have been developed, and used f or studies of the intramolecular and intermolecular DNA ligation react ions of topoisomerase II. Intramolecular ligation occurred only to sin gle-stranded acceptor molecules with the ability to base-pair to the d onor 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 intermolecul ar 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 r educed efficiency, as compared with the base-pairing control. Experime nts demonstrated that the enzyme can ligate DNA molecules, where nucle otides were either removed or inserted in the employed acceptor molecu les. The results indicate that topoisomerase II might be responsible f or similar rearrangements in vivo, since gapped and nicked DNA structu res appear as intermediates in processes such as replication and repai r. The reaction is, however, likely to be constrained by the requireme nt of base-pairing for ligation.