MODULATION OF DNA TOPOISOMERASE-I ACTIVITY BY P53

The tumor suppressor protein p53 plays a central role in the cellular response to genotoxic lesions and has been shown to be activated by mo st anticancer agents such as mitomycin C. We here show that mitomycin C treatment of human MCF7 breast adenocarcinoma cells results in incre ased topoisomerase I activity as measured by relaxation of supercoiled DNA and by phosphorylation of SR protein splicing factor. The increas e in catalytic activity occurs in parallel with the nuclear accumulati on of p53, resulting in detectable activation of topoisomerase I withi n less than 1 h of drug treatment. Furthermore, topoisomerase I co-imm unoprecipitates with nuclear p53, suggesting that the activation of to poisomerase I may be a result of a direct interaction between the two proteins. In vitro experiments with purified recombinant proteins show that p53 increases the catalytic activities of topoisomerase I as mea sured by relaxation of supercoiled DNA, stabilization of the covalent topoisomerase I-DNA complex (in the presence of camptothecin), and pho sphorylation of SR protein splicing factor ASF/SF2. Furthermore, topoi somerase I sediments at a higher molecular weight in the presence of p 53 as revealed by sucrose density gradient analysis in the absence of DNA. Finally, p53 modifies the thermal stability of topoisomerase I, p rotecting it from heat denaturation. Taken together, our results show that topoisomerase I and p53 form molecular complexes in vitro as in v ivo, and we suggest that the p53-mediated response to DNA damage may, at least in part, involve activation of topoisomerase I.