Abstract
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.
Highlights
These experiments indicate that helices at anenzyme-bound DNA crossing were often thought binding of yeast DNA topoisomerase I1 to DNA cross- to consist of a DNA segment containing the DNA gate, and a DNA segment to be transported through ing Mg(II), and that this mode of binding strongly the gate
Whereasstabilization of DNA crossovers by eukaryotic DNA topoisomerase I1 is sufficient for the retention of supercoils in thepresence of excess vaccinia virus DNA topoisomerase, the AMPPNP-driven formation of knotted DNAby eukaryotic DNA topoisomerase I1 requires the enzyme to serve a dualrole of stabilizing the crossovers and transporting one DNA segment through anotherA. lthough it is conceivable that knotting may result from the topological trapping of nodes in a supercoiled DNA by the AMPPNP-driven action of a single type I1 enzyme molecule, a number of studies suggest that knotformation is much enhanced inthe presence of ligands that bridge pairs of DNA segments (Liu et al, 1976, 1980; Hsieh, 1983; Wasserman and Cozzarelli, 1991; Annan et al, 1992)
The modes of interactionbetween DNA and yeast DNA topoisomerase I1 were probed by linking number measurements of plasmid DNA relaxed by vaccinia virus DNA topoisomerase in the absence and presence of stoichiometric amounts of yeast DNA topoisomerase 11, and by knotting of DNA rings upon addition of AMPPNP to mixtures of DNA and yeast DNA topoisomerase 11
Summary
Vol 268, No 19, Issue of July 5, pp. 14250-14255,1993 Printed in U.S.A. On the Simultaneous Binding of Eukaryotic DNA Topoisomerase I1 to a Pair of Double-Stranded DNA Helices*. In the second type of Because the transport of one double-stranded DNAsegexperiments, formation of knotted DNA rings by the ment through another by a type I1 DNA topoisomerase necaddition of AMPPNP to mixtures of yeast DNA topoi- essarily requires the juxtaposition of two DNA segments at somerase I1 and different topological forms of DNA some stage of the enzyme’s catalytic cycle, the pair of DNA ringswas studied. The efficiency of DNA trans- 3.1-kb plasmid by two-dimensional agarose gel electrophoport by the bound yeast enzyme following the addition of resis, blot-hybridization, and autoradiography.N , nicked rings; AMPPNP, and knot formation accompanying suacphrocess, (-), negatively supercoiled plasmid; R, plasmid after relaxation by were examined.
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