DNA supercoiling is an important determinant of variety of molecular processes because of its influence on substrate properties of DNA. Many proteins that bind DNA interact in a negatively supercoil dependent manner. The higher free energy associated with negative supercoiling is utilized for binding and subsequent transactions. Protein components of DNA replication, segregation, transcription and recombination machinery interact with DNA in topology dependent manner. Topoisomerases, thus play essential role in DNA transaction processes. Owing to their influence on diverse cellular functions, the enzymes are considered as global regulators. Movement of macromolecular assemblies along the DNA helix generates internal torsional strain during replication and transcription. This strain manifests itself as local domains of supercoils ahead and behind the helix tracking machinery. If left unchecked, accumulation of these supercoils could severely affect the survival of the cells. Cellular systems thus employ more than one topoisomerase, which act in concert to regulate DNA topology and maintain a steady-state level of supercoiling necessary for integrity and functionality of DNA molecules.