Direct observation of helicase–topoisomerase coupling within reverse gyrase
Open Access
- 5 May 2020
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 117 (20), 10856-10864
- https://doi.org/10.1073/pnas.1921848117
Abstract
Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.Keywords
Funding Information
- Agence Nationale de la Recherche (ANR-19-CE11-0001)
- Ligue Contre le Cancer (NA)
- Centre National de la Recherche Scientifique (NA)
- Université Paris Diderot (NA)
- École Normale Supérieure (NA)
- Institut National de la Santé et de la Recherche Médicale (NA)
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