Mechanistic Studies of Type IA and IIA Topoisomerases Using Orthogonal Single-Molecule Techniques

Kathryn Harris Gunn

doi:https://doi.org/10.21985/N2ST19

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Mechanistic Studies of Type IA and IIA Topoisomerases Using Orthogonal Single-Molecule Techniques

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Topoisomerases are ubiquitous enzymes involved in maintaining the supercoiled state of DNA in the cell. Structural, biophysical, and biochemical studies have provided an overall view of the mechanisms of DNA transformation by these enzymes, but many aspects, particularly their dynamic characteristics, remain poorly understood. Type IA topoisomerases change the topology of DNA using an enzyme-bridged strand passage mechanism, where one DNA strand is passed through a transient break in the second strand. Recently, single-molecule Magnetic Tweezer (MT) experiments, which monitor the topology of a single DNA molecule in real time, revealed that an important feature of the type IA mechanism is the presence of pauses between relaxation events. However, MT experiments alone cannot determine whether the protein remains bound to the DNA during the pauses in relaxation or the relationship between conformational movements in the protein and topological changes in the DNA. In order to concurrently monitor DNA topology and protein conformational changes, we combined two orthogonal single-molecule techniques, MT and total internal reflection fluorescence (TIRF) microscopy. Using MT we were able to monitor a paramagnetic bead attached to a strand of DNA. The height of the bead provided information about the topological state of the DNA. Concurrently we used protein induced fluorescence enhancement (PIFE) to monitor protein binding to the DNA and domain movements in the protein. This novel single-molecule combination of MT and PIFE allowed us to observe that successful DNA strand passage events by type IA topoisomerases correlate with domain movements in the protein. Specifically, domain III of Escherichia coli topoisomerase I (EcTopoI) moves toward the protein to open a gate for the capture of ssDNA; this movement toward the protein had not previously been hypothesized. Furthermore, we found that the protein remains bound to DNA during pauses in relaxation, but that there are domain movements in the protein during pauses. From this we inferred that the enzyme is constantly changing conformation and attempting to change the topology of DNA, but only succeeding in a fraction of the attempts. The mechanism can therefore be described as a series of attempts to pass one DNA strand through a break in another strand, which generates the pauses, culminating in a successful relaxation event. Using the MT instrument developed for the combined MT-TIRF microscope, we also experimentally demonstrated the theoretically predicted presence of a discontinuous buckling transition for two intertwined or braided DNA tethers. These tethers mimic catenations in DNA, which are a common byproduct of DNA replication in the cell and often a substrate for topoisomerases. Our experiments also showed that when two braided DNA molecules buckle, they do not form a single plectoneme domain, but multiple smaller domains, which may have implications for protein binding and activity. These studies have provided a better understanding of the mechanism of type IA topoisomerases and validated the theoretical understanding for catenated DNA substrates. The multiple-attempt mechanism uncovered for type IA topoisomerases, provides a fascinating look at a molecular machine, which separates chemical events, single stranded DNA (ssDNA) cleavage and domain movement, from mechanical steps, ssDNA strand passage and relaxation. It is likely that other enzymes, including type II topoisomerases, may employ a similar multiple-attempt mechanism

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