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 eukar yotic 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 o f the DNA products were measured to quantitate supercoils that were co nstrained by the stoichiometrically bound yeast enzyme molecules; in p arallel, the same yeast enzyme-supercoiled DNA mixtures were incubated with a nonhydrolyzable ATP analog AMPPNP (adenosine 5'-(beta,gamma-im ido)triphosphate) instead of the vaccinia enzyme, and DNA linking numb er changes following the addition of AMPPNP were measured to monitor D NA transport mediated by the yeast enzyme and AMPPNP. In the second ty pe of experiments, formation of knotted DNA rings by the addition of A MPPNP to mixtures of yeast DNA topoisomerase II and different topologi cal forms of DNA rings was studied. These experiments indicate that bi nding of yeast DNA topoisomerase II to DNA crossings is significant, e specially in low salt media containing Mg(II), and that this mode of b inding strongly affects DNA knotting. It appears, however, that stabil ization of DNA crossovers by the eukaryotic type II enzyme is not dire ctly related to its DNA transport activity.