ON THE SIMULTANEOUS BINDING OF EUKARYOTIC DNA TOPOISOMERASE-II TO A PAIR OF DOUBLE-STRANDED DNA HELICES

Stabilization of crossings of pairs of DNA helices by binding of eukaryotic DNA topoisomerase II was studied by two types of experiments. In one, mixtures of yeast DNA topoisomerase II and supercoiled DNA were incubated with vaccinia virus topoisomerase, and the linking numbers of the DNA products were measured to quantitate supercoils that were constrained by the stoichiometrically bound yeast enzyme molecules; in parallel, the same yeast enzyme-supercoiled DNA mixtures were incubated with a nonhydrolyzable ATP analog AMPPNP (adenosine 5'-(beta,gamma-imido)triphosphate) instead of the vaccinia enzyme, and DNA linking number changes following the addition of AMPPNP were measured to monitor DNA transport mediated by the yeast enzyme and AMPPNP. In the second type of experiments, formation of knotted DNA rings by the addition of AMPPNP to mixtures of yeast DNA topoisomerase II and different topological forms of DNA rings was studied. These experiments indicate that binding of yeast DNA topoisomerase II to DNA crossings is significant, especially in low salt media containing Mg(II), and that this mode of binding strongly affects DNA knotting. It appears, however, that stabilization of DNA crossovers by the eukaryotic type II enzyme is not directly related to its DNA transport activity.