The tumor suppressor protein p53 stimulates the formation of the human topoisomerase I double cleavage complex in vitro Kent Søe 1 , Hella Hartmann 1 , Bernhard Schlott 1 , Tinna Stevnsner 2 and Frank Grosse* ,1 1 Institute of Molecular Biotechnology, Department of Biochemistry, Beutenbergstrasse 11, D-07745 Jena, Germany; 2 Danish Center for Molecular Gerontology, Department of Molecular and Structural Biology, University of Aarhus, C.F. Møllers Alle´ Bldg. 130, DK-8000 Aarhus C, Denmark Previous studies have shown that human topoisomerase I interacts directly with the tumor-suppressor protein p53. In the past few years it has repeatedly been suggested that topoisomerase I and p53 may play a joint role in the response to genotoxic stress. This led to the suggestion that p53 and human topoisomerase I may cooperate in the process of DNA repair and/or apoptosis. Recently we have demonstrated that a human topoisomerase I cleavage complex can be recognized by an additional topoisomerase I molecule and thereby form a so-called double cleavage complex. The double cleavage complex creates an about 13 nucleotides long single-stranded gap that may provide an entry site for recombinational repair events. Here we demonstrate that p53 stimulates both the DNA relaxation activity as well as the formation of the human topoisomerase I double cleavage complex by at least a factor of six. Stimulation of topoisomerase I activity by p53 is mediated via the central part of topoisomerase I. We also show that human, bovine, and murine p53 stimulate human topoisomerase I relaxation activity equally well. From these results it is conceivable that p53’s stimulatory activity on topoisomerase I may play a role in DNA recombination and repair as well as in apoptosis. Oncogene (2002) 21, 6614 – 6623. doi:10.1038/sj.onc. 1205912 Keywords: topoisomerase I; p53; DNA damage; repair; recombination; genomic instability Introduction Human topoisomerase I (htopoI) is a nuclear enzyme which is involved in several important pathways, such as transcription and replication (Yang et al., 1987; Stewart and Schutz, 1987; Snapka et al., 1988; Stewart et al., 1990), where the removal of positive supercoils is required for ongoing RNA and DNA synthesis. HtopoI can remove positive as well as negative supercoils by catalyzing a reversible transesterification reaction and thereby forming a covalent 3’-phospho- tyrosyl bond between one DNA strand and an enzyme tyrosine residue while leaving a free 5’-hydroxyl terminus next to the cleavage site (Andersen et al., 1996). A family of alkaloids called camptothecins (CPT), which possess an antitumor effect, specifically inhibit the reversion of this reaction due to a specific interaction with htopoI. This leads to the stabilization of a reversible covalent complex between htopoI and the DNA backbone, a so-called ‘cleavage complex’, thereby introducing a long-lived nick into the DNA. The cytotoxic effect of these types of complexes is most likely due to a fragmentation of the genome that occurs when ongoing replication forks collide with covalently bound topoisomerases (Ryan et al., 1991; Squires et al., 1991). Different DNA lesions have been found to induce reversible htopoI cleavage complexes. These lesions include abasic sites, oxidative DNA damages, DNA alkylation or arylation events, gaps, base mismatches, and UV-photoproducts (Lanza et al., 1996; Pourquier et al., 1997a,b, 1999, 2001; Christiansen and Wester- gaard, 1999). Also, the nucleoside analog 1-b-D- arabinofuranosylcytosine (Ara-C), a drug used for the treatment of acute leukemia, has been shown to stabilize the covalent complex (Pourquier et al., 2000). All the aforementioned lesions cause a local distortion of the DNA double helix and this is believed to promote the binding of these sites by htopoI and/or to slow down religation. UV lesions, DNA methyla- tion, and Ara-C have also been shown to stabilize htopoI cleavage complexes in vivo (Subramanian et al., 1998; Mao et al., 2000; Pourquier et al., 2000, 2001). When such htopoI-DNA complexes are not repaired they may lead to genomic instability, e.g. through illegitimate recombination and block of transcription and/or replication (Ryan et al., 1991; Tsao et al., 1993; Megonigal et al., 1997; Wu and Liu, 1997). Mao et al. (2000) found that in a wt-p53 cell line htopoI cleavage complexes formed in vivo upon UV damage whereas these complexes did not form in a cell line carrying a p53 point mutation on amino acid 175. The tumor suppressor gene p53 has been found to contain mutations in more than 50% of all human tumors (Vogelstein and Kinzler, 1992). p53 is involved in regulatory processes of cell-cycle progression and in cellular responses following DNA damage (Lane, 1992). After exposure to stress signals p53 is activated Received 27 February 2002; revised 24 July 2002; accepted 25 July 2002 *Correspondence: F Grosse; E-mail: fgrosse@imb-jena.de Oncogene (2002) 21, 6614 – 6623 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc