The human prostate cancer cell line DU145 and its CPT-resistant variant DU145/RC and the human ovarian cancer cell line 2774 and its CPT-resistant variant 2774/RC were kindly provided by Beppino Giovanella (Stehlin Foundation for Cancer Research, Houston, Tex.). The human lymphoblast cell line RPMI 8402 and its CPT-resistant variant CPT-K5 were obtained from Toshiwo Andoh (Soka University, Tokyo, Japan). Chin (The Cancer Institute of New Jersey). The human breast cancer cell line ZR-75-1 was kindly provided by K.-V. Monkey kidney fibroblast BSC cells and Chinese hamster lung V79 cells were obtained from the American Type Culture Collection (Manassas, Va.). These results are consistent with a model in which arrest of the elongating RNA polymerase complexes by Top I-DNA covalent complexes triggers 26S proteasome-mediated degradation of Top I and subsequent repair of the exposed single-strand breaks. Transcription recovery is dependent on both degradation of Top I and functional transcription-coupled repair (TCR).

In the present study, we show that Top I-DNA covalent complexes arrest transcription and trigger transcription-dependent degradation of both Top I and the large subunit of RNA polymerase II (RNA Pol II 0).

It has been suggested that degradation of Top I in the Top I-DNA covalent complex represents a potential repair mechanism for Top I-DNA covalent complexes ( 9). Recently, CPT has been demonstrated to specifically induce degradation of Top I via a ubiquitin-26S proteasome pathway ( 8, 9). Repair of Top I-DNA covalent complexes is conceptually challenging because of the reversibility of the complexes and the bulkiness of the protein-DNA adducts. Indeed, studies with a cell-free simian virus 40 DNA replication system have suggested that collisions between the replication forks and Top I-DNA covalent complexes result in irreversible arrest of the fork, the formation of double-strand DNA breaks, and the conversion of reversible Top I-DNA complexes into Top I-linked DNA breaks ( 6, 13, 29). The induction of these DNA damage responses by CPT is consistent with the notion that Top I-DNA covalent complexes are converted into DNA damage by their collisions with the replication forks. In addition to lethality, the collision between the DNA replication fork and the Top I-DNA covalent complex is responsible for G 2 cell cycle arrest, activation of NF-κB and ATR (for “AT mutated and Rad3 related”), stabilization of p53, increased accumulation of c- fos and c- jun mRNAs, and phosphorylation of Chk1 and RPA (reviewed in reference 16). Studies using CPT have demonstrated that moving DNA replication forks can process Top I-DNA covalent complexes into potentially lethal DNA lesions, which explains the S-phase-specific cytotoxicity of CPT ( 6). Because of the reversible nature of Top I-DNA covalent complexes, lethality occurs only upon their irreversible conversion to DNA strand breaks ( 17). CPT, the prototypic Top I poison, is known to block the religation reaction of Top I, resulting in accumulation of Top I-DNA covalent complexes ( 12, 23). In the complex, Top I is covalently linked to the 3′-phosphoryl end of the transient single-strand DNA break via a tyrosyl phosphate bond (reviewed in reference 17). They represent the covalent intermediates of the cleavage-religation reaction of Top I ( 12). Top I-DNA covalent complexes are reversible ( 21, 23). Despite the increasing importance of Top I-DNA covalent complexes, their repair mechanism(s) remains largely unclear. In addition to Top I-directed antitumor drugs, many DNA lesions (e.g., UV adducts, 1-β- d-arabinofuranosylcytosine-substituted DNA, benzopyrene-DNA adducts, and 8-oxo-guanine-modified DNA) are now known to trap Top I-DNA covalent complexes to form secondary DNA lesions ( 16, 22, 32). The antitumor drug camptothecin (CPT) is the first agent demonstrated to induce Top I-DNA covalent complexes, which is believed to be exclusively responsible for the antitumor activity of CPT ( 5). Topoisomerase I (Top I)-DNA covalent complexes, more commonly referred to as TopI cleavable or cleavage complexes, represent a unique type of DNA lesion ( 12, 21, 23).

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