Regulation of the catalytic cycle of topoisomerase II

Topoisomerase II is an essential enzyme that modulates DNA topology in vivo. The catalytic cycle of this enzyme can be dissected into at least six discrete steps: (1) enzyme$\cdot$DNA binding; (2) pre-strand passage DNA cleavage/religation; (3) DNA strand passage; (4) post-strand passage DNA cleavage/religation; (5) ATP hydrolysis; and (6) enzyme turnover. Each activity catalyzed by topoisomerase II has the potential to be a control point for the regulation of overall enzyme activity. The present work demonstrates that the initial topoisomerase II-mediated DNA cleavage event is regulated by the enzyme's interaction with its DNA substrate. The enzyme requires that both the cleavage helix and the passage helix be present in the enzyme$\cdot$DNA complex prior to DNA cleavage. In addition, results demonstrate that the catalytic activity of topoisomerase II is altered by the presence of cyclobutane pyrimidine dimers in substrate DNA. A novel non-turnover DNA catenation assay was developed to demonstrate that these DNA lesions inhibit overall catalytic activity by decreasing the ability of the enzyme to carry out its DNA strand passage event. Finally, phosphorylation stimulates the catalytic activity of topoisomerase II specifically by enhancing the ATP hydrolysis step of the catalytic cycle. Phosphorylation mediated by either casein kinase II or protein kinase C enhances overall enzyme activity by the same mechanism.

All of these studies examine the individual steps of the catalytic cycle of topoisomerase II to determine the exact mechanism by which effectors alter enzyme activity. These represent the first studies that employ this approach to determine specific control points within the catalytic cycle of the enzyme. With the development of an assay for the DNA strand passage event of topoisomerase II, it is now possible to examine each step of the catalytic cycle that precedes enzyme turnover.