Reverse gyrase is the only enzyme known to introduce positive supercoils into DNA. Positive supercoiling is achieved by the functional cooperation of a helicase-like and a topoisomerase domain. The isolated helicase-like domain is a DNA-stimulated ATPase, and the isolated topoisomerase domain can relax supercoiled DNA. In the context of reverse gyrase, these individual activities are suppressed or attenuated. The helicase-like domain of Thermotoga maritima reverse gyrase is a nucleotide-dependent conformational switch that binds DNA and ATP cooperatively. It provides a nucleotide-dependent DNA-binding site to reverse gyrase and thus serves as a valuable model for the investigation of the effect of nucleotides on DNA processing by reverse gyrase that is key to its supercoiling activity. To improve our understanding of the structural basis for the functional cooperation of a helicase domain with a DNA topoisomerase, we have determined the structures of the isolated helicase-like domain of T. maritima reverse gyrase in five different conformations. Comparison of these structures reveals extensive domain flexibility in the absence of conformational restrictions by the topoisomerase that is consistent with single-molecule Förster resonance energy transfer experiments presented here. The structure of the first ADP-bound form provides novel details about nucleotide binding to reverse gyrase. It demonstrates that reverse gyrases use the canonical nucleotide binding mode common to superfamily 2 helicases despite large deviations in the conserved motifs. A characteristic insert region adopts drastically different structures in different reverse gyrases. Counterparts of this insert region are located at very different positions in other DNA-processing enzymes but may point toward a general role in DNA strand separation.
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http://dx.doi.org/10.1021/bi200236a | DOI Listing |
bioRxiv
October 2024
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland, 21205, USA.
DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively.
View Article and Find Full Text PDFInt J Syst Evol Microbiol
October 2024
Institute for Extra-cutting-edge Science and Technology Avant-garde Research (X-star), Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Japan.
A novel mesophilic bacterium, strain SS33, was isolated from a deep-sea hydrothermal vent chimney at Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean. The cells of strain SS33 were motile short rods with a single polar flagellum. The growth of strain SS33 was observed at the temperature range between 33 and 55 °C (optimum growth at 45 °C), at the pH range between 5.
View Article and Find Full Text PDFJ Mol Evol
October 2024
Institut Pasteur, 25 rue du Docteur Roux, 75015, Paris, France.
The existence of LUCA in the distant past is the logical consequence of the binary mechanism of cell division. The biosphere in which LUCA and contemporaries were living was the product of a long cellular evolution from the origin of life to the second age of the RNA world. A parsimonious scenario suggests that the molecular fabric of LUCA was much simpler than those of modern organisms, explaining why the evolutionary tempo was faster at the time of LUCA than it was during the diversification of the three domains.
View Article and Find Full Text PDFJ Mol Evol
October 2024
Departamento de Ingeniería Genética, Cinvestav Unidad Irapuato, Km 9.6 Libramiento Norte Carretera Irapuato-León CP. 36824, Irapuato, Gto., Mexico.
The ultimate consequence of Darwin's theory of common descent implies that all life on earth descends ultimately from a common ancestor. Biochemistry and molecular biology now provide sufficient evidence of shared ancestry of all extant life forms. However, the nature of the Last Universal Common Ancestor (LUCA) has been a topic of much debate over the years.
View Article and Find Full Text PDFMicrobes Environ
June 2024
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University.
All cells must maintain the structural and functional integrity of the genome under a wide range of environments. High temperatures pose a formidable challenge to cells by denaturing the DNA double helix, causing chemical damage to DNA, and increasing the random thermal motion of chromosomes. Thermophiles, predominantly classified as bacteria or archaea, exhibit an exceptional capacity to mitigate these detrimental effects and prosper under extreme thermal conditions, with some species tolerating temperatures higher than 100°C.
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