DNA knots are deleterious for living cells if not removed. Several theoretical and simulation approaches address the question of how topoisomerases select the intermolecular passages that preferentially lead to unknotting rather than to the knotting of randomly fluctuating DNA molecules, but the formation of knots in vivo remains poorly understood. DNA knots form in vivo in non-replicating and replicating molecules, and supercoiling as well as intertwining are thought to play a crucial role in both the formation and resolution of DNA knots by topoisomerase IV. To confirm this idea, we used two-dimensional agarose gel electrophoresis run with different concentrations of chloroquine to demonstrate that non-replicating pBR322 plasmids grown in a topoisomerase I-defective E. coli strain (RS2λ) were more negatively supercoiled than in a wild-type strain (W3110) and, concurrently, showed significantly fewer knots. In this way, using wild-type and E. coli mutant strains, we confirmed that one of the biological functions of DNA supercoiling is to reduce the formation of DNA knots.
