The p53 tumor suppressor stimulates the catalytic activity of human topoisomerase IIα by enhancing the rate of ATP hydrolysis

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 IIα, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated ~2-3-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 IIα- 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 IIα and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIα 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 IIα by ICRF- 193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIα was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIα 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 IIα. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIα.