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bioRxiv · 10.1101/696609

Cryo-EM structure of the complete E. coli DNA Gyrase nucleoprotein complex

Vanden Broeck, A.·Ortiz, J.·Lamour, V.
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Abstract

DNA Gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, the detailed architecture of DNA Gyrase from the model genetic organism E. coli, is still missing, impeding structure-function analysis of E. coli-specific catalytic regulation and limiting the study of conformational intermediates of this highly flexible macromolecule. Herein, we determined the complete molecular structure of the E. coli DNA Gyrase bound to a 180 bp DNA and the antibiotic Gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil with unprecedented details the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of closed and pre-opening states of the DNA-binding domain provides a better understanding of the allosteric movements of the enzyme complex. In this region, the local atomic resolution reaching up to 3.0 [A] enables the identification of the antibiotic density in the DNA complex. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates. The type 2A DNA topoisomerases (Top2) are nanomachines that control DNA topology during multiple cellular processes such as replication, transcription and cell division 1-4. These enzymes catalyze the transport of a DNA duplex through a double strand break to perform DNA relaxation, decatenation and unknotting. DNA Gyrase plays a vital role in the compaction of the bacterial genome and is the sole type 2 topoisomerase able to introduce negative supercoils into DNA, a reaction coupled to ATP hydrolysis 5.

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Vanden Broeck, A., Ortiz, J., Lamour, V.. 2019-07-11. Cryo-EM structure of the complete E. coli DNA Gyrase nucleoprotein complex. https://doi.org/10.1101/696609

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