Reverse gyrase is the only topoisomerase known till date that can introduce positive supercoils into DNA. From the biochemical and biophysical evidence gained so far, it is known that the enzyme only relaxes negatively supercoiled DNA, and introduces positive supercoils in presence of ATP at high temperatures. The exact picture of conformational changes and inter-domain cross talk during the positive supercoiling reaction has not yet been captured biophysically. The proposed work is aimed at gaining insight into the mechanism of positive supercoiling by reverse gyrase from T.maritima and at identifying the determinants that governs this reaction. Single molecule FRET studies will be undertaken to understand how the different domains of the enzyme communicate with each other and with DNA to facilitate positive supercoiling. Real time binding studies will be carried out to understand the mode and stages of DNA binding and nucleotide binding to the enzyme. Potential DNA binding sites will be identified on the enzyme and mutants will be generated for further binding studies. This work will be the first attempt to capture the “enzyme in action” and monitor the conformational changes during positive supercoiling reaction. Achieving the specific aims of this ambitious project will significantly improve our understanding about the dynamics of the protein and the DNA during the positive supercoiling reaction and hence will unravel the underlying mechanism.