DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase II␣, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated ϳ2؊3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase II␣-mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase II␣ and DNA. The effects of p53 on the catalytic inhibition of topoisomerase II␣ were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase II␣ by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase II␣ was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase II␣ to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase II␣. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase II␣.Eukaryotic DNA topoisomerase II is a nuclear enzyme that modulates the topological states of DNA via transient doublestrand breaks in DNA coupled with subsequent strand passage step (1-4). The mechanism of topoisomerase II activity involves DNA cleavage, strand passage, and religation, succeeded by enzyme turnover, a process requiring ATP hydrolysis (5, 6). During this cycle, the enzyme covalently binds to DNA forming an intermediate called topoisomerase II-DNA covalent cleavable complex (1-4). Topoisomerase II is essential for cell viability (7,8) and has been implicated in many important cellular processes such as replication, transcription, recombination, and chromosomal segregation (7-10). The enzyme also functions as a major structural component of mitotic chromosome and interphase nuclear scaffolds (11,12).Topoisomerase II is the intracellular target for a variety of active agents currently used in the treatment of human cancers (1, 13-15). By stabilizing the covalent enzyme-associated...