Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 20, Pages 10856-10864Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1921848117
Keywords
DNA topoisomerase; helicase; single molecule; magnetic tweezers
Categories
Funding
- Ligue Nationale Contre le Cancer Equipe Labellisee program
- French Agence Nationale de la Recherche (ANR) [ANR-19-CE11-0001]
- Institut Jacques Monod, University of Paris Diderot (University of Paris)
- INSERM
- CNRS
- Ecole Normale Superieure (Paris Sciences et Lettres)
- Agence Nationale de la Recherche (ANR) [ANR-19-CE11-0001] Funding Source: Agence Nationale de la Recherche (ANR)
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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 similar to 2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds similar to 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.
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