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

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 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.