Single-molecule analysis of DNA uncoiling by a type II topoisomerase

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment(1). This enables them to untangle DNA(2-6) and relax the interwound supercoils (plectonemes) that arise in twisted DNA(7). In vivo, they are r esponsible for untangling replicated chromosomes and their absence at mitos is or meiosis ultimately causes cell death(8,9). Here we describe a microma nipulation experiment in which we follow in real time a single Drosophila m elanogaster topoisomerase II acting on a linear DNA molecule which is mecha nically stretched and supercoiled(10-13). By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoi ls during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the appl ied stress. Finally, in the absence of ATP, we observe the clamping of a DN A crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.