Antitumour drugs impede DNA uncoiling by topoisomerase I

Abstract

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.

Hyperphosphorylation of RNA Polymerase II in Response to Topoisomerase I Cleavage Complexes and Its Association with Transcription- and BRCA1-dependent Degradation of Topoisomerase I

“…44 However, CPT seems to preferentially inhibit Top1-mediated relaxation of positive versus negative DNA supercoiling. 14,45 Finally, CPT may also interfere with RNA splicing by blocking the SR-kinase activity of Top1, 46 which would promote the formation of R-loops 47 and therefore also impair transcriptional elongation. 44,48 Our study (Fig.…”

Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

“…To accurately measure the number of stand passages per enzyme binding event, i.e. the processivity, which could be much larger than the total writhe in the DNA molecule, we generated an "infinite" supercoiled substrate (34). As supercoils were removed, the accompanying increase in the height of the bead was recorded in real time (Fig.…”

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements

“…Since their invention (28)(29)(30), MT techniques have often been used to analyze the interactions between DNA and DNA ligands, including small drug molecules (25,31,32) and proteins (33)(34)(35)(36)(37)(38). MT can be used to apply controlled force and torsion to single DNA molecules and enables measurement of the influence of such nanomechanical stresses on the DNA structure, primarily through the assessment of end-to-end distance at nanometric resolution.…”