Gyrase . DNA complexes visualized as looped structures by electron microscopy.

Abstract

Gyrase bound to duplex DNA in the absence of ATP is seen by electron microscopy as a nearly spherical particle frequently located at the intersection of two duplex DNA strands. Such looped structures with gyrase situated at the base of the loops are observed with both linear and circular DNA substrates, and two or three individual DNA molecules bound to the same protein are also seen at high DNA concentrations. Addition of the nonhydrolyzable beta,gamma-imido analog of ATP to the gyrase . DNA reaction mixture prior to sample fixation for microscopy reduces the frequency of gyrase molecules found at DNA intersections. Looped structures similar to those of the gyrase . DNA complex are also seen with the complex of DNA and the A subunit of gyrase. When negatively supercoiled DNA which has been partially relaxed by gyrase in the absence of ATP is fixed for electron microscopic examination, intermediate forms are observed that contain both supercoiled and relaxed loops in a single DNA molecule, with the enzyme located at the common base of the loops. These results suggest that gyrase possesses multiple DNA-binding sites, a feature which allows the enzyme to hold DNA in constrained loops. The relation of these observations to the mechanism of gyrase action is discussed.