| Criteria | Type I Topoisomerases | Type II Topoisomerases | Remarks |
| Strand Cleavage | Cleave one strand of the DNA duplex | Cleave both strands of the DNA duplex | Key mechanistic difference; impacts energy requirement and function |
| ATP Requirement | Do not require ATP | Require ATP hydrolysis for activity | Type II enzymes use ATP to drive strand passage |
| Mechanism of Action | Relieve supercoiling by creating a transient single-strand break | Relieve supercoiling and untangle knots by double-strand passage | Type II can perform more complex topological changes |
| Effect on Supercoiling | Typically relax negative supercoils | Can relax both negative and positive supercoils | Type II enzymes are more versatile in topological stress management |
| Examples | Topoisomerase I (E. coli), Top1 (human) | DNA gyrase, Topoisomerase IV (E. coli); Top2α/β (human) | Gyrase introduces negative supercoils—a unique feature |
| Subunit Composition | Usually monomeric | Typically multimeric (e.g., dimer or tetramer) | Structural complexity correlates with function |
| Biological Roles | Maintain DNA topology during replication and transcription | Decatenation of daughter chromosomes, DNA supercoiling, unknotting | Type II essential in mitosis and bacterial DNA replication |
