Mechanism of negative supercoil relaxation by Topoisomerase IV

Type II topoisomerases are essential enzymes that alter DNA topology by making a transient double-stranded break in one segment of the DNA and passing another DNA segment through the break such that the sign of the crossing DNA segments is inverted. DNA gyrase has served as a paradigm for mechanistic studies of topoisomerase action. Prescient biochemistry predicting a positive sign for the crossing DNA segments during DNA gyrase supercoiling activity has recently been confirmed by cryo-electron microscope studies of the enzyme bound to DNA. These studies demonstrated a central role of the C-terminal domain of the GyrA subunit in shaping the DNA crossover. We show biochemically that exchanging the DNA GyrA CTD for the homologous one from the ParC subunit of Topoisomerase IV alters the orientation of the DNA crossover from a positive one to a negative one. The sign of the pre-catalytic DNA crossover in Topoisomerase IV is also negative. We suggest that relaxation of negatively supercoiled DNA by Topoisomerase IV proceeds by the opposite of the mechanism by which DNA gyrase supercoils DNA.