Title: Camptothecin binding to topoisomerase I-DNA complexes impedes DNA uncoiling and induces the accumulation of positive supercoils
Abstract: 5735 DNA topoisomerase I (Top1) catalyzes the relaxation of positively or negatively supercoiled DNA via the formation of a transient Top1-DNA covalent intermediate, which acts as a swivel to allows DNA uncoiling or rewinding, respectively. Camptothecin targets Top1 by reversibly stabilizing this covalent complex. During S-phase, the collision of advancing replication forks with Top1-DNA-drug complexes are thought to induce the formation of potentially lethal DNA lesions that cause cell death. Several camptothecin analogs, including irinotecan and topotecan have significant activity against adult and pediatric solid tumors. A detailed understanding of Top1-drug interactions is critical for the optimal clinical development of these chemotherapeutics; however, the dynamic interactions underlying Top1 poisoning by camptothecins and their biological ramifications remain largely unknown. Employing a single-molecule technique allowed us to monitor the binding and unbinding of an individual topotecan molecule in real-time and quantify the drug-induced trapping of Top1 on DNA. In these experiments, a single double-stranded DNA molecule is anchored between a glass surface and a paramagnetic bead. A pair of magnets generates an upward stretching force, while rotating the magnets about their axis mechanically injects positive or negative supercoils. By continuously monitoring bead height (DNA extension), the degree of DNA supercoiling is determined in real-time. Our studies show that a bound topotecan molecule alters the Top1 swivel in two ways. First, we conclude that an individual topotecan traps Top1 for at least 2 minutes, an increase of roughly a factor of 100 in comparison to when topotecan is absent. Second, our findings also show that topotecan significantly hinders DNA rotation within the covalent complex. Unexpectedly, however, this effect was more pronounced on the removal of positive (overwound) rather than negative supercoils. These single-molecule observations led us to posit that this bias towards negative supercoil removal would also be induced by camptothecin in vivo, during cellular processes such as transcription and replication that induce DNA supercoiling. Indeed, yeast as a model system, we show that camptothecin poisoning of Top1 induces the accumulation of positive supercoils in G1 and S-phases of the cell cycle, independent of Topoisomerase II. These findings suggest a novel mechanism for drug-induced cell death. Instead of the covalent complex presenting a physical barrier to the advancing fork, the local domain of positive supercoils generated ahead of the fork, which is not efficiently removed by the drug-bound Top1, may itself hamper fork progression. The resultant stalling of the replication machinery would result in fork collapse and the formation of potentially lethal DNA lesions that ultimately induce cell death.
Publication Year: 2007
Publication Date: 2007-05-01
Language: en
Type: article
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