DNA topoisomerase I is a nuclear enzyme involved in transcription, recombination, and DNA damage recognition. Previous studies have shown that topoisomerase I interacts directly with the tumor-suppressor protein p53. p53 is a transcription factor that activates certain genes through binding to specific DNA sequences. We now report that topoisomerase I can be stimulated by both latent and activated wild-type p53 as well as by several mutant and truncated p53 proteins in vitro, indicating that sequence-specific DNAbinding and stimulation of topoisomerase I are distinct properties of p53. These assays also suggest that the binding site for topoisomerase I on p53 is between amino acids 302 and 321. In living cells, the interaction between p53 and topoisomerase I is strongly dependent on p53 status. In MCF-7 cells, which have wild-type p53, the association between the two proteins is tightly regulated in a spatial and temporal manner and takes place only during brief periods of genotoxic stress. In marked contrast, the two proteins are constitutively associated in HT-29 cells, which have mutant p53. These findings have important implications for both cellular stress response and genomic stability, given the ability of topoisomerase I to recognize DNA lesions as well as to cause illegitimate recombination.DNA topoisomerase I is a nuclear enzyme essential for most aspects of nucleic acid metabolism (1). The ability of topoisomerase I to relax supercoiled DNA through transient single-stranded DNA cleavage, strand passage, and religation is needed during transcription to relieve superhelical stress (2, 3). In addition, topoisomerase I serves as a modulator of transcriptional initiation through physical interaction with transcription factors such as the TATA box-binding protein, which is a constituent of the general transcription factor IID (TFIID) complex (4, 5). This property does not require DNA cleavage but is based on protein-protein interactions. Recent results show that topoisomerase I also possesses a proteinkinase activity, which is specific for serine residues of splicing factors containing an Arg-Ser motif. Phosphorylation of these splicing factors is believed to influence gene expression by altering the splice pattern (6, 7).Eukaryotic topoisomerase I is structurally, functionally, and evolutionarily related to site-specific recombinases (8). During catalysis, topoisomerase I forms a covalent DNA-protein complex with the 3Ј end of one DNA strand. This reaction intermediate can religate with either the 5Ј hydroxyl end of the cleaved strand or with a 5Ј hydroxyl end of a heterologous DNA molecule, resulting in recombinant DNA (9, 10). In yeast, increased topoisomerase I activity is accompanied by increased levels of illegitimate recombination, whereas treatment of mammalian cells with camptothecin and other topoisomerase I-directed antitumor drugs leads to sister chromatid exchange and chromosomal aberrations (11,12). This is most likely because camptothecin treatment results in accumulation of covalent ...
The tumor suppressor protein p53 plays a central role in the cellular response to genotoxic lesions and has been shown to be activated by most anticancer agents such as mitomycin C. We here show that mitomycin C treatment of human MCF7 breast adenocarcinoma cells results in increased topoisomerase I activity as measured by relaxation of supercoiled DNA and by phosphorylation of SR protein splicing factor. The increase in catalytic activity occurs in parallel with the nuclear accumulation of p53, resulting in detectable activation of topoisomerase I within less than 1 h of drug treatment. Furthermore, topoisomerase I co-immunoprecipitates with nuclear p53, suggesting that the activation of topoisomerase 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 measured by relaxation of supercoiled DNA, stabilization of the covalent topoisomerase I-DNA complex (in the presence of camptothecin), and phosphorylation of SR protein splicing factor ASF/SF2. Furthermore, topoisomerase I sediments at a higher molecular weight in the presence of p53 as revealed by sucrose density gradient analysis in the absence of DNA. Finally, p53 modifies the thermal stability of topoisomerase I, protecting it from heat denaturation. Taken together, our results show that topoisomerase I and p53 form molecular complexes in vitro as in vivo, and we suggest that the p53-mediated response to DNA damage may, at least in part, involve activation of topoisomerase I.
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