DNA topoisomerases are nature's tools for resolving the unique problems of DNA entanglement that occur owing to unwinding and rewinding of the DNA helix during replication, transcription, recombination, repair, and chromatin remodeling. These enzymes perform topological transformations by providing a transient DNA break, formed by a covalent adduct with the enzyme, through which strand passage can occur. The active site tyrosine is responsible for initiating two transesterifications to cleave and then religate the DNA backbone. The cleavage reaction intermediate is exploited by cytotoxic agents, which have important applications as antibiotics and anticancer drugs. The reactions mediated by these enzymes can also be regulated by their binding partners; one example is a DNA helicase capable of modulating the directionality of strand passage, enabling important functions like reannealing denatured DNA and resolving recombination intermediates. In this review, we cover recent advances in mechanistic insights into topoisomerases and their various cellular functions.