Baker Matthew L, Johnson Haley R, Eckerty Ryan A, Fogg Jonathan M, Summers Silvia L, Vayssières Marlène, Marechal Nils, Lamour Valérie, Olson Wilma K, Zechiedrich Lynn
Department of Biochemistry & Molecular Biology, University of Texas Health Sciences Center at Houston, Houston, TX USA.
Graduate Program in Quantitative & Computational Biosciences, Baylor College of Medicine, Houston, TX, USA.
Res Sq. 2025 Aug 18:rs.3.rs-7265879. doi: 10.21203/rs.3.rs-7265879/v1.
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 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 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 β-pinwheel of gyrase. For the other structure, in which the negatively supercoiled minicircle wrapped a positive supercoil around a β-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.
细菌gyrase是II型拓扑异构酶中独一无二的，它能将负超螺旋引入DNA。由于该过程的复杂性以及难以对其进行可视化，gyrase的作用机制细节仍有待阐明。具体而言，gyrase位点选择过程中碱基序列、局部DNA可变形性和整体DNA拓扑结构之间的相互作用尚不清楚。为了了解gyrase如何与DNA相互作用并选择作用位点，我们创建了一种基于形状的识别方法，以从低温电子显微镜密度中确定DNA序列，将其作为一串嘌呤和嘧啶，我们最终将其与我们之前两个结合了gyrase的负超螺旋DNA结构中的DNA进行了匹配。在这两种结构中，将被gyrase切割的DNA螺旋（门或G段）映射到小环中回文序列的每一侧，DNA（相对于酶）的方向相反。对于一种结构，G段序列是小环中最灵活的序列之一，促进了观察到的DNA弯曲。侧翼序列高度不灵活，这可能阻止了其围绕gyrase的β-风车结构缠绕。对于另一种结构，其中负超螺旋小环围绕gyrase的β-风车结构缠绕了一个正超螺旋，G段仅包含平均可变形性的碱基对步。这项工作突出了作用位点周围的碱基序列和局部可变形性如何加速DNA缠绕，以促进gyrase的负超螺旋活性。它进一步证明了从冷冻电镜结构中识别与蛋白质相互作用的DNA序列的实用性。
