Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T-segment) through a transient double-strand break in a second segment of DNA (gate or G-segment) in an ATP-dependent reaction. simplification activity. We found that topoisomerase IV yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases. INTRODUCTION Topoisomerases are enzymes that modify and regulate the topology of cellular DNA (1-5). The regulation of DNA topology is essential in all organisms since vital cell processes such as DNA replication and transcription give rise to a myriad of topological problems including knotting catenation and excess supercoiling of genomic DNA. Type I topoisomerases cut a single strand of DNA through an FTY720 ATP-independent mechanism that allows for the relaxation of supercoils and in some cases passage of duplex DNA through a nick (2). Type II topoisomerases on the other hand cut both strands of one segment of DNA (gate or G-segment) and pass a second segment (transfer or T-segment) through the transient double-strand break via an ATP-dependent mechanism (Figure 1A) (1-3 5 This two-gate mechanism results in unidirectional strand passage (6 7 Though they share a similar core strand passage mechanism the FTY720 type II topoisomerase subclasses type IIA and type IIB are structurally biochemically and evolutionarily distinct (8-10). FTY720 Type IIA topoisomerases are capable of relaxing and introducing supercoils as well as generating and removing intramolecular and intermolecular links i.e. knots and catenanes. Type IIA topoisomerases play a fundamental role in chromosome segregation during cell division by unlinking catenated sister chromatids thus enabling daughter cells to receive the proper complement of chromosomes (3). They are also likely required to reduce the level of DNA knotting that is expected in highly compacted DNA which would have deleterious consequences if allowed to accumulate (11-14). Though type IIA topoisomerases facilitate these critical unlinking and unknotting processes via a strand-passage mechanism random strand-passage would not lead to specific and complete unlinking and Nrp1 unknotting (15). Rather it would result in a dynamic equilibrium between linking and unlinking reactions. Figure 1. The FTY720 interaction of type IIA topoisomerases with DNA. (A) Core strand-passage mechanism for type IIA topoisomerases [adapted from Bates and Maxwell (7)]. (1) Type IIA topoisomerase (blue yellow and orange) binds duplex G-segment DNA (green). ( … Rybenkov (15) showed that non-supercoiling type IIA topoisomerases (i.e. type IIA topoisomerases with the exception of DNA gyrase which negatively supercoils DNA) simplify the global topology of DNA shifting it away from an equilibrium distribution of catenanes knots and supercoils toward a less entangled topology. An equilibrium distribution referred to as topological equilibrium of knots catenanes and supercoils is achieved if every encounter between two DNA segments has an equal probability of resulting in strand passage. Because type I topoisomerases do not consume energy they shift DNA topology toward equilibrium. In contrast type IIA topoisomerases can shift global topology away from equilibrium which implies that they couple topology sensing to strand passage to achieve preferential simplification of knots catenanes and supercoils. Since type IIA topoisomerases consume ATP thermodynamic principles are not violated but the mechanism by which the energy of ATP hydrolysis is coupled to topology simplification remains elusive (16). Specifically it is unclear how an enzyme that acts on the scale of nanometers FTY720 is able to assess the global topology of DNA. If the enzyme only interacts with a single DNA crossing how is it able to determine that a strand passage event would result in the removal rather than the creation of a linkage (Figure 1B)? Several mechanistic models of topology simplification by type IIA topoisomerases have been proposed (15 17 though to date the.