Antitumour drugs impede DNA uncoiling by topoisomerase I.
Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.
Koster
Daniel A
DA
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Palle
Komaraiah
K
Bot
Elisa S M
ES
Bjornsti
Mary-Ann
MA
Dekker
Nynke H
NH
D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008280:Magnetics; D036103:Nanotechnology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D059004:Topoisomerase I Inhibitors; D019772:Topotecan / Q000494:pharmacology*
D004278:DNA, Superhelical; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D019772:Topotecan; D004264:DNA Topoisomerases, Type I