Mechanism of ATP-dependent DNA supercoiling, relaxation and decatenation by type IIA DNA topoisomerases

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Topoisomerases are enzymes that regulate the degree of DNA supercoiling. Members of the type IIA subfamily catalyze topological changes by strand passage: Both strands of the DNA substrate are cleaved, mediated by two catalytic tyrosines, and a second double-stranded DNA segment is passed through the gap. The enzymes share a two-fold symmetric structure with three interfaces, termed N-gate, DNA-gate and C-gate, whose coordinated opening and closing enables strand passage. Despite their common core architecture, type IIA topoisomerases catalyze different hallmark reactions: ATP-dependent DNA supercoiling (gyrase), ATP-dependent DNA relaxation (topo II), or ATP-dependent DNA decatenation (topo IV). In single-molecule FRET experiments with gyrase, we have revealed a cascade of DNA- and nucleotide-induced conformational changes at the beginning of the supercoiling reaction, but have never observed the strand-passage event. Gyrase with a single catalytic tyrosine catalyzes DNA supercoiling in the absence of strand passage by a nicking/closing mechanism. This reaction might be a backup mechanism or a general pathway. ATP-dependent DNA relaxation (topo II) and decatenation (topo IV) require two tyrosines and strand passage. The conformational changes in the catalytic cycles of topo II and topo IV and their temporal correlation are currently unclear.Topo II and topo IV can catalyze DNA relaxation and decatenation in vitro, whereas gyrase supercoils and decatenates DNA. In bacteria, topo IV is responsible for the decatenation of replication intermediates, gyrase for the removal of positive supercoils ahead of the replication fork. Some bacteria contain only one type IIA topoisomerase, which needs to catalyze both reactions. Species-specific insertions in the common gyrase scaffold modulate the enzymatic properties of gyrases, and may affect the balance between supercoiling and decatenation activities. To comprehensively understand the mechanistic spectrum employed by type IIA topoisomerases, we propose to investigate the mechanism of ATP-dependent DNA supercoiling and decatenation by different gyrases, and of ATP-dependent DNA relaxation and decatenation by topo II and topo IV. Specifically, we will delineate DNA- and ATP-dependent conformational changes of these enzymes during the different reactions, and analyze the temporal coordination of these conformational changes in single-molecule FRET experiments. We will dissect the enzymatic activities provided by the gyrase scaffold and their modulation by species-specific insertions, as well as the role of these insertions for the balance of supercoiling and decatenation activities. Overall, the results will reveal the determinants for the balance between different topological changes catalyzed by the same enzyme, as well as differences and similarities between the same topological change, but catalyzed by different enzymes.