DNA deformability defines sequence-dependent capture of E. coli gyrase

DNA deformability defines sequence-dependent capture of E. coli gyrase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article DNA deformability defines sequence-dependent capture of E. coli gyrase Lynn Zechiedrich, Matthew Baker, Haley Johnson, Ryan Eckerty, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7265879/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Bacterial gyrase, unique among type II topoisomerases, introduces negative supercoils into DNA. Mechanistic details of gyrase still must be elucidated because of the complexity of the process and the difficulty in visualizing it. Specifically, the interplay among base sequence, local DNA deformability, and global DNA topology for gyrase site selection is unclear. To understand how gyrase interacts with DNA and selects a site of action, we created an ad hoc shape-based recognition methodology to ascertain DNA sequence from cryogenic electron microscopy densities as a string of purines and pyrimidines, which we conclusively matched to the DNA in our two previous structures of negatively supercoiled DNA bound to E. coli gyrase. The DNA helices to be cleaved by gyrase (the Gate or G-segments) in both structures mapped to each side of a palindrome in the minicircle, with the DNA (relative to the enzyme) in opposite orientations. For one structure, the G-segment sequence was among the most flexible in the minicircle, facilitating the observed bend in the DNA. The flanking sequence was highly inflexible, which presumably prevented wrapping about the beta-pinwheel of gyrase. For the other structure, in which the negatively supercoiled minicircle wrapped a positive supercoil around a beta-pinwheel of gyrase, the G-segment contained base-pair steps of only average deformability. This work highlights how base sequence and local deformability around the site of action expedite DNA wrapping to facilitate the negative supercoiling activity of gyrase. It further demonstrates the utility of identifying protein-interacting DNA sequences from cryo-EM structures. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biophysics/Computational biophysics Biological sciences/Biochemistry/DNA DNA supercoiling DNA minicircle DNA site recognition DNA base-pair step deformability Type II topoisomerase Full Text Additional Declarations Yes there is potential Competing Interest. J.M.F. and L.Z. are co-inventors on several patents covering the minicircle technology and are shareholders in Twister Biotech, Inc. (rebranded as Velvet Therapeutics, Inc.). The authors declare no other competing interests. Supplementary Files NCOMMS256005softwareinfo.zip Software_infromation Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7265879","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":500708994,"identity":"7ad5da72-aadb-4d7b-8b91-3028d3151377","order_by":0,"name":"Lynn 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Mechanistic details of gyrase still must be elucidated because of the complexity of the process and the difficulty in visualizing it. Specifically, the interplay among base sequence, local DNA deformability, and global DNA topology for gyrase site selection is unclear. To understand how gyrase interacts with DNA and selects a site of action, we created an ad hoc shape-based recognition methodology to ascertain DNA sequence from cryogenic electron microscopy densities as a string of purines and pyrimidines, which we conclusively matched to the DNA in our two previous structures of negatively supercoiled DNA bound to E. coli gyrase. The DNA helices to be cleaved by gyrase (the Gate or G-segments) in both structures mapped to each side of a palindrome in the minicircle, with the DNA (relative to the enzyme) in opposite orientations. For one structure, the G-segment sequence was among the most flexible in the minicircle, facilitating the observed bend in the DNA. 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