VACCINIA DNA TOPOISOMERASE-I - EVIDENCE SUPPORTING A FREE ROTATION MECHANISM FOR DNA SUPERCOIL RELAXATION

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand c leavage and religation by forming a transient phosphotyrosyl linkage b etween the DNA and Tyr-274, resulting in the release of DNA supercoils . For type I topoisomerases, two mechanisms have been proposed for sup ercoil release: (1) a coupled mechanism termed strand passage, in whic h a single supercoil is removed per cleavage/religation cycle, resulti ng in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple super coils are removed per cleavage/religation cycle, resulting in few inte rmediates and early product formation. To determine the mechanism, sin gle-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a singl e site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversio n were determined by kinetic simulation. Few intermediates and early p roduct formation were observed. From these data, the rate constants fo r cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilib rium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, w hile the rotation rate of the cleaved DNA strand is fast (similar to 2 0 rotations/s). Thus, the average number of supercoils removed for eac h cleavage event greatly exceeds unity (Delta n = 5) and depends on ki netic competition between religation and supercoil release, establishi ng a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.